JP2005535604A - Polymer nano-articles containing therapeutic agents - Google Patents

Abstract

A hydrophilic polymer article containing a polymer scaffold covalently bound to one or more therapeutic agents (eg, a drug or drug conjugate molecule). The article optionally further comprises a recognition element (RE) for binding to biomolecular structures expressed in specific cells or specific tissues to facilitate targeting and / or delivery. And can be covalently attached to it. The present invention relates to the field of therapeutic entities. More particularly, the present invention relates to polymeric articles that incorporate bioactive ingredients.

Description

(Field of Invention)
The present invention relates to the field of therapeutic entities. More particularly, the present invention relates to polymeric articles that incorporate bioactive ingredients.

(Background of the Invention)
Small molecule drugs often contain a substantially hydrophobic structure with limited water solubility and blood solubility. These compounds interact strongly with the biological membrane and diffuse rapidly through the biological membrane, thus easily exiting the bloodstream and entering the tissue non-specifically. Such undesired distributions can result in high dose requirements, rapid clearance, and toxic side effects. In fact, small molecule drugs can present toxicity issues whether they are hydrophobic or hydrophilic in nature. In order to improve performance, drugs are water-soluble synthetics, for example, to change biodistribution, reduce toxicity, reduce multiple drug resistance (MDR) efflux, and prolong activity Polymers (eg, N- (2-hydroxypropyl) methacrylamide (HPMA)) (Burtles et al., Hum. Exp. Toxicol. 1988, 17, 93-104); natural polymers (eg, dextran; and proteins (eg, albumin) (Schutte MT et al., Crit. Rev. Ther. Drug Carrier System. 1999, 16, 245-288) The size and solubility of the drug compared to the drug conjugate. Provides a basis for understanding the difference: binding to hydrophilic polymers This results in larger molecules with reduced dispersibility and increased water solubility and blood solubility, thus non-specific transmembrane is significantly reduced, thus such drug-polymer conjugates are Compared to alone, tissues that exist longer in the bloodstream and thus have leaky vessels can be passively targeted.

  Active targeting of a drug is intended to localize a bioactive agent to a specific type of cell or cell component, tissue type or tissue component, or organ with a specific biomolecular structure (eg, a specific This has been achieved through the use of ligand constructs that can bind to receptors that are overexpressed in cancer cells. For example, HPMA-drug conjugates are further functionalized with monoclonal antibodies that target receptors that are overexpressed in certain cancers. In another example, the drug molecule is directly conjugated to a monoclonal antibody (mAb). For example, Seattle Genetics (Seattle, WA) has the compound SGN15 (which is a mAb functionalized with doxorubicin (dox) and binds to a surface Ley antigen expressed in specific tumors) .

  Other materials and methods that improve performance beyond the current state of the art (eg, via improved therapeutic efficacy, larger therapeutic index, improved solubility, or lower dosage regimen) are important to the field It is a great addition.

(Summary of the Invention)
As used herein, the terms “article” and “nanoarticle” are used interchangeably.

  The present invention relates to a hydrophilic polymer nano-article comprising: i) a scaffold comprising a crosslinked hydrophilic building block; and ii) one or more therapeutic agents (eg, a drug or drug) covalently bound to the scaffold. Conjugated molecule). The nanoarticle can further include one or more recognition elements (RE), as appropriate, to facilitate targeting and / or delivery. The nanoarticle can also consist of polyethylene glycol (PEG) based molecules, if desired. The PEG chain can be attached to the surface of the nanoarticle at one end and functionalized with RE at the other end to serve as a linker or tether, and / or the PEG chain can be other useful or desired The features can be provided in this hydrophilic nanoarticle. The present invention further relates to methods of synthesizing these nanoarticles and various applications in which these nanoarticles can be used.

  The nanoarticles of the present invention preferably have a diameter of about 5 nm to about 100 nm, more preferably about 10 nm to about 70 nm. The size of the nanoarticles allows their use as bioactive entities in mammals. In order to avoid uptake by the reticuloendothelial system, the nano article is preferably less than 100 nm. In order to avoid renal clearance, the nanoarticle is preferably larger than 5 nm. Reverse microemulsion polymerization can yield a scaffold for the nanoarticles of the present invention.

  The therapeutic agent is incorporated into the nanoarticle via a covalent bond to the hydrogel scaffold. A preferred therapeutic entity is doxorubicin or a doxorubicin derivative.

  The RE binds the nanoarticle of the invention to a desired biomolecule that is overexpressed or otherwise found on specific cell surfaces or specific tissues. The number of REs per nanoarticle can range from 2 to about 1000, preferably from 2 to 500. The nano-article can further comprise one or more types of RE, if desired. As used herein, a “type” of RE is defined as an RE of a particular molecular structure. A further advantage of the present invention is that multiple RE systems with complementary features can be incorporated into a single nanoarticle.

  The nano-article scaffold includes a cross-linked hydrophilic building block. This building block is crosslinked in the dispersed aqueous phase of the inverse microemulsion. Carbohydrates and carbohydrate derivatives are preferably used as building blocks.

(Detailed description of the invention)
As used herein and in the appended claims, the terms “a” and “an” mean “one or more”, unless otherwise indicated.

  “Water soluble” means in this specification and in the appended claims having a solubility in water of greater than 10 mg / mL, and preferably greater than 50 mg / mL.

  The nanoarticles of the present invention comprise two types of molecular structures: scaffolds and therapeutic agents. In presently preferred embodiments, the nanoarticle also includes a third molecular structure: RE, which binds to a biomolecule expressed in a specific cell or a specific tissue. The RE and therapeutic agent are covalently bound to the nanoarticle scaffold. As used herein, the terms “nanoarticle scaffold”, “hydrogel scaffold” and “scaffold” are used interchangeably and the bioactive agent and recognition of the nanoarticle (polymer matrix structure). This refers to the part formed before the elements are joined. The scaffold of the present invention is a chemically cross-linked nanoscopic hydrogel structure.

  Nanoarticles are hydrophilic and are intended for use in mammals. In one embodiment, each nanoarticle is functionalized with two or more recognition elements, which recognition elements possess a high affinity for a biomolecular target, and these recognition elements are a polymer of the nanoarticle. Covalently bonded to the matrix structure. The invention further relates to a method of synthesizing these polymer nanoarticles.

  Nano articles of the present invention can range in size from about 5 nm to about 1000 nm, more preferably from about 5 nm to about 100 nm, and most preferably from about 10 nm to about 70 nm. Nanoarticles in the 10-70 nm size range can effectively avoid renal clearance and uptake by the reticuloendothelial cell line. Further, such nanoarticles can advantageously exit the bloodstream to reach the desired cell, tissue, or organ target.

  The nanoarticles of the present invention can be advantageously used in particular in the treatment of cancer. The leaky vasculature found in tumors may allow these nanoarticles to leave the bloodstream and be concentrated within the tumor. This effect (described as enhanced permeability and retention (EPR) for macromolecular drugs) was observed to be universal in solid tumors (Maeda H. et al., J. Controlled Release, 2000, 65). , P271-284). The main mechanism for the EPR effect on macromolecules is retention, while low molecular weight substances are not retained and are returned to the circulating blood by diffusion. Thus, nano-articles with a diameter of 5-100 nm can accumulate in solid tumors. Thus, the nanoarticles of the present invention are naturally concentrated in the tumor, even before target binding, providing greater efficacy and less systemic toxicity.

  Hydrophilic building blocks with polymerizable groups are used to form hydrogel scaffolds. This building block is crosslinked in the dispersed aqueous phase of the inverse microemulsion. The number of polymerizable groups attached to a single building block can range, for example, from about 1 to 3 for low molecular weight building blocks and 10 or more for polymer building blocks. Building blocks containing more than one polymerizable machine can act as crosslinkers and are capable of forming hydrogel scaffolds. Formation of different compliance hydrogels during polymerization by using different amounts and proportions of building blocks than a set of building blocks having one, two, or more polymerizable groups Is possible.

  Exemplary polymerizable groups include acrylate moieties, acrylamide moieties, methacrylate moieties, methacrylamide moieties, vinyl ether moieties, styryl moieties, epoxide moieties, maleic acid derivative moieties, diene moieties, substituted diene moieties, thiol moieties, alcohol moieties, amines. Part, hydroxyamine part, carboxylic acid part, carboxylic acid anhydride part, carboxylic acid halide part, aldehyde part, ketone part, isocyanate part, succinimide part, carboxylic acid hydrazide part, glycidyl ether part, siloxane part, alkoxysilane part, Alkyne moiety, azide moiety, 2'-pyridyldithiol moiety, phenylglyoxal moiety, iodo moiety, maleimide moiety, imide ester moiety, dibromopropionate moiety, and iodo Cetyl portions, but not limited to.

  Free radical polymerization: Preferred polymerizable functional groups are acrylate moieties, acrylamide moieties, methacrylate moieties, and methacrylamide moieties. Such portions are susceptible to free radical polymerization. Free radical polymerization can be easily accomplished via UV light and photoinitiators, redox-coupled free radical polymerization initiators, or a combination of heat and heat activated initiators.

  The building blocks that can be used to form the scaffold of the article include small molecules having one polymerizable group or multiple polymerizable moieties, which can act as low molecular crosslinkers. Exemplary building blocks include acrylamide, sodium acrylate (NaA), diacetone acrylamide (DAA), malonate acrylamide (MalAc), levulinic acid acrylamide, methylene bisacrylamide, 2,2-bisacrylamide ammonium acetate, 2- Acrylamide glycolic acid, 2-aminoethyl methacrylate, N- (3-aminopropyl) methacrylamide (APMA), ornithine monoacrylamide, ornithine diacrylamide sodium salt, N-acryloxytris (hydroxymethyl) -methylamine, hydroxyethyl acrylate N- (2-hydroxypropyl) methacrylamide, N- (2-hydroxypropyl) acrylamide, 2-sulfoethyl methacrylate, 2-meta Ryloxyethyl glucoside, glucose monoacrylate, glucose-1- (N-methyl) acrylamide, glucose-2-acrylamide, glucose-1,2-diacrylamide, maltose-1-acrylamide, sorbitol monoacrylate, sorbitol diacrylate, sucrose Diacrylate, sucrose mono (ethylenediamine acrylamide), sucrose di (ethylenediamine acrylamide), sucrose di (diethylenetriamine acrylamide), kanamycin tetraacrylamide, kanamycin diacrylamide, sucrose mono (ethylenediamine acrylamide) mono (diethylenetriamine acrylamide) mono (phenylalanine) sodium salt, And other acrylate derivatized sugars The acrylamide derivatized sugar.

  The building blocks are selected to achieve the desired content of specific functional groups in the article scaffold. Such functional groups can improve solubility and can also be used as a point of attachment between the therapeutic agent and the RE. For example, APMA can be used to introduce amines, sodium acrylate can be used to introduce carboxylates, and DAA can be used to incorporate ketones. In a preferred embodiment, at least some of the building blocks are N, N'-cystine bisacrylamide (CiBA) monomers, which have the following formula I:

ＣｉＢＡは、以下の反応スキームに従って、Ｌ−シスチン（ＩＩ）を２当量のアクリロイルクロリド（ＩＩＩ）と反応させることによって、調製され得る：

CiBA can be prepared by reacting L-cystine (II) with 2 equivalents of acryloyl chloride (III) according to the following reaction scheme:

ＣｉＢＡは、水溶性であり、そして他の足場構築ブロック（例えば、アクリレート官能基化炭水化物）を重合させて、ヒドロゲル足場を形成し得る。さらに、そのジスルフィド結合は、還元後、リンカーの結合のための遊離チオールを提供する。

CiBA is water soluble and other scaffold building blocks (eg, acrylate functionalized carbohydrates) can be polymerized to form hydrogel scaffolds. Furthermore, the disulfide bond provides a free thiol for linker attachment after reduction.

  In preferred embodiments, at least some of the building blocks are carbohydrates. In the case of a carbohydrate building block, this carbohydrate region contains a plurality of hydroxyl groups, wherein at least one hydroxyl group is modified to contain at least one polymerizable group.

  The carbohydrate region of the carbohydrate building block can include carbohydrates or carbohydrate derivatives. For example, the carbohydrate region can be a simple sugar (eg, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, neuraminic acid, galacturonic acid, glucuronic acid, ioduronic acid, glucose, ribose, arabinose. , Xylose, lyxose, allose, altrose, apiose, mannose, gulose, idose, galactose, fucose, fructose, fructofuranose, rhamnose, arabinofuranose, and talose); disaccharides (eg, maltose, sucrose, Lactose or trehalose); trisaccharides; polysaccharides (eg, cellulose, starch, glycogen, alginic acid, inulin, pullulan, dextran, sulfate Stran, chitosan, glycosaminoglycan, heparin, heparin sulfate, hyaluronic acid, gum tragacanth, xanthan, other carboxylic acid-containing carbohydrates, uronic acid-containing carbohydrates, lactulose, arabinogalactans, and derivatives thereof, and any of these Mixtures); or derived from modified polysaccharides. Other representative carbohydrates include sorbitan, sorbitol, chitosan, and glucosamine. Carbohydrates can contain amine groups in addition to hydroxyl groups, and the amine groups or hydroxyl groups can be modified (ie, substituted) to contain bridging groups, other functional groups, or combinations thereof.

  Carbohydrate-based building blocks can be prepared from carbohydrate precursors (eg, sucrose, inulin, dextran, pullulan, etc.) by coupling techniques known in the field of bioorganic chemistry (eg, G Hermanson, Bioconjugation Techniques, Academic Press, San Diego, 1996, pages 27-40, 155, pages 183-185, pages 615-617; and S. Hanesian, Preparative Carbohydrate Chemistry, Marcel Dekker, New York, 1997). For example, the crosslinkable group can be attached to the carbohydrate by dropping acryloyl chloride onto the amine functionalized sugar. Amine functionalized sugars can be prepared by reaction of ethylenediamine (or other amines) with 1,1'-carbonyldiimidazole activated sugars. Ester-linked reactive groups can be synthesized by reacting acrylic or methacrylic anhydride with the hydroxyl group of a carbohydrate (eg, inulin) in pyridine.

  Carbohydrate-based building blocks can also be prepared by partial (or complete) functionalization of carbohydrates with moieties that are known to polymerize under free radical conditions. For example, methacrylic esters can be placed on carbohydrates at various substitution levels by reaction of the carbohydrate with methacrylic anhydride or glycidyl methacrylate (Vervoort L. et al., International Journal of Pharmaceuticals, 1998, 172, 127-135).

  In presently preferred embodiments, at least some of the building blocks are inulin multimethacrylate (IMMA) monomers, which have the following formula IV:

式ＩＶにおけるｎの値は、約５〜約５０である。現在好ましい実施形態において、約１０〜約２０の平均重合度（ＤＯＰ）を有するイヌリンが使用される。従って、好ましい実施形態において、ｎは、約８〜約１８である。イヌリンがメタクリレート部分で官能基化される程度、すなわち、イヌリン上の、メタクリル酸エステルに変換されてＩＭＭＡを生成するヒドロキシル部分の数は、イヌリンおよびメタクリル酸無水物の出発物質の濃度および重量比によって支配される、統計学的プロセスである。官能基化の程度は、１〜１００の単糖類繰り返し単位ごとに１つのメタクリレート、より好ましくは、３〜２０の単糖類繰り返し単位ごとに１個のメタクリレートの範囲であり得る。イヌリンへのエステル結合は、インビボでの分解の部位として有利に機能し得、物品が分解して身体から除去されることを可能にする。デキストランマルチメタクリルアミドおよびプルランマルチメタクリルアミドは、類似の方法を使用して調製され得る、さらなる好ましい構築ブロックである。

The value of n in Formula IV is about 5 to about 50. In presently preferred embodiments, inulin having an average degree of polymerization (DOP) of about 10 to about 20 is used. Thus, in a preferred embodiment, n is about 8 to about 18. The degree to which inulin is functionalized with a methacrylate moiety, ie, the number of hydroxyl moieties on inulin that are converted to methacrylic esters to produce IMMA, depends on the concentration and weight ratio of the inulin and methacrylic anhydride starting materials. It is a controlled statistical process. The degree of functionalization can range from 1 methacrylate per 1-100 monosaccharide repeat units, more preferably 1 methacrylate per 3-20 monosaccharide repeat units. The ester linkage to inulin can advantageously function as a site for degradation in vivo, allowing the article to degrade and be removed from the body. Dextran multimethacrylamide and pullulan multimethacrylamide are additional preferred building blocks that can be prepared using similar methods.

  Carbohydrate-based building blocks can also be obtained by chemoenzymatic methods (Martin BD et al., Macromolecules, 1992, 25, 7081) (eg, where Pseudomonas cepacia is a monosaccharide acetate in pyridine). It can be prepared by catalyzing transesterification with vinyl) or by direct addition of acrylates (Pilesky S. et al., Macromolecules, 1999, 32, 633-636). Because many derivatized carbohydrates are known to those skilled in the art of carbohydrate chemistry, other functional groups may be present.

  Carbohydrate structures are selected in part with respect to their hydrophilicity. Nanoarticles that incorporate a substantially hydrophobic drug must have a very hydrophilic scaffold so that high water solubility is maintained after functionalization with the drug. The nano-article of the present invention composed of IMMA has a high water content. “High moisture content” as used herein and in the appended claims refers to about 65 to about 98 wt% water, more preferably about 75 to about 98 wt% water, most preferably Means an article composed of about 80-97 wt% water. Accordingly, the amount of degradation products is less than articles having higher polymer concentrations. High water content scaffolds can also reduce immunogenicity because there are fewer interacting surfaces for immune system components.

  In addition to carbohydrate-based building blocks, other examples of acrylate-derivatized polymer building blocks or acrylamide-derivatized polymer building blocks include polyethylene glycol-based molecules having molecular weights ranging from 200 to 40,000 daltons (eg, polyethylene Glycol diacrylate).

  In a preferred embodiment, a degradable linkage is included in the cross-linked scaffold to facilitate metabolism of the hydrogel scaffold and thereby facilitate drug release in the desired time frame. Degradable linkages are included, among other things, through the use of polylactide, polyglycolide, poly (lactide-co-glycolide), polyphosphadine, polyphosphate, polycarbonate, polyamino acid, polyanhydride, and polyorthoester based building blocks. obtain. In addition, degradable linkages can be used to attach the polymerizable moiety to the carbohydrate. For example, IMMA includes an ester moiety that connects the inulin carbohydrate backbone formed during the free radical polymerization used to produce the scaffolds of the present invention to an alkyl chain. In addition, low molecular crosslinkers that contain similar hydrolyzable moieties as polymers (eg, carbonates, esters, urethanes, orthoesters, amides, and phosphoric acids) can be used as building blocks. In order to function as a degradable component in a hydrogel scaffold, these building blocks must be functionalized with two or more polymerizable moieties. For example, polyglycolide diacrylate, polyorthoester diacrylate, and acrylate substituted polyphosphazenes, acrylate substituted polyamino acids, or acrylate substituted polyphosphate polymers can be used as degradable building blocks. A methacrylate or acrylamide moiety can be used in place of the acrylate moiety in the above example. Similarly, small molecules comprising hydrolyzable segments and two or more acrylates, methacrylates, or acrylamides can be used. Such degradable polymers and small molecule building blocks can be functionalized with acrylates, methacrylates, acrylamides or similar moieties by methods known in the art.

  The nanoarticle scaffolds and scaffold degradation products of the present invention are designed to be non-toxic and excluded from the body. They can be degradable and preferably have a carbohydrate-based, polyamino acid-based, polyester-based, or PEG-based core and are controlled by sugar identity, crosslink density, and other characteristics. Has a degradation rate. Thus, the article can be metabolized in the body and prevents unwanted accumulation in the body.

  Chemoselective polymerization: Chemoselective building blocks can also be used to form scaffolds. A typical example of this strategy may be the use of polysaccharides that are partially oxidized to contain multiple aldehydes in a reverse microemulsion. Di (amino-oxy) containing compounds (eg, those made by reacting ethylenediamine with NHS ester of Boc-amino-oxyacetic acid (see reaction scheme below)) are then amino-oxy It can be used as a cross-linking agent through the reaction of an oxidized sugar aldehyde that reacts with a functional group.

（逆ミクロエマルジョンにおける物品足場製作）：本発明の物品は、まず、逆ミクロエマルジョンの分散水相中に可溶化した親水性構築ブロックの架橋を介してナノ規模のヒドロゲル足場を形成することにより、製作される。有機溶媒および非反応性界面活性剤は、重合後に除去されて、架橋された水溶性ナノ範囲物品を生じる。

Article scaffold fabrication in reverse microemulsion: Articles of the invention are first formed by forming nanoscale hydrogel scaffolds via cross-linking of hydrophilic building blocks solubilized in the dispersed aqueous phase of the reverse microemulsion. Produced. The organic solvent and non-reactive surfactant are removed after polymerization to yield a crosslinked water-soluble nano-range article.

  Inverse microemulsions for scaffold fabrication are surfactant stabilized aqueous nano-particles dispersed in a continuous oil phase combining aqueous buffer or water, building blocks, organic solvents, surfactants and initiators in appropriate ratios. It is formed by obtaining a stable phase of the droplet. Stable inverse microemulsion formulations can be found by those skilled in the art using known methods. They are described, for example, in Microemulsion Systems (edited by HL Rosano and M. Clausse), New York, N .; Y. : M. Dekker, 1987; and Handbook of Microemulsion Science and Technology (edited by P. Kumar and KL Mittel), New York, N .; Y. : M. Discussed in Dekker, 1999. In the present invention, an aqueous phase having a solubilized hydrophilic building block is added to an organic solvent containing one or more solubilized surfactants to form a reverse microemulsion.

  The dispersed aqueous phase comprises hydrophilic building blocks solubilized at about 5 to about 65 wt%, preferably about 5 to about 25 wt%, most preferably 10 to 20 wt%. While not wishing to be bound by theory, the use of high water content hydrogel scaffolds may also reduce immunogenicity in the end user. This is because there are few foreign surfaces recognized by immune system components. High water content also provides compliance through a more flexible scaffold. Thus, when binding to a cell surface receptor, the article can conform to the cell surface and bind more surface receptors. Binding more receptors may allow the article to function better as an antagonist. Furthermore, without wishing to be bound by theory, it is believed that the article cell surface cover can inhibit the signaling pathways of other cells.

  The polymerization of the building blocks in the nanodroplets of the dispersed aqueous phase of the inverse microemulsion follows procedures known to those skilled in the art (eg, ODG GG; Principles of Polymerization, 3rd edition, Wiley, New York, 1991; L H. Spelling, Introduction to Physical Polymer Science, Chapter 1, pages 1-21, John Wiley and Sons, New York, 1986; and R. B. Seymour and C. E. Carrher, 11th. Chapter, pp. 193-356, Dekker, New York, 1981). Polymerization takes place in the dispersed phase of microemulsions and inverse microemulsions (for review, see Antonietti, M .; and Basten, R., Macromol. Chem. Phys. 1995, 196, 441; reverse micro (See Holtzscherer, C .; and Candau, F., Colloids and Surfaces, 1988, 29, 411) for a study of the polymerization of hydrophilic monomers in the dispersed aqueous phase of the emulsion). Such polymerization can result in articles in the 5 nm to 50 nm size range.

The size of the nanodroplets in the dispersed aqueous phase is determined by the relative amounts of water, surfactant and oil phase used. Surfactants are utilized to stabilize the inverse microemulsion. These surfactants do not contain crosslinkable moieties; they are not building blocks. Surfactants that can be used include commercially available surfactants such as: Aerosol OT (AOT), polyethyleneoxy (n) nonylphenol (Igepal ^™ , Rhodia Inc. Surfactants and Specialties, Cranbrook, NJ), Sorbitan esters (Sorbitan monooleate (Span® 80), Sorbitan monolaurate (Span® 20), Sorbitan monopalmitate (Span® 40), Sorbitan monostearate (Span®) (Trademark) 60), sorbitan trioleate (Span (R) 85), and sorbitan tristearate (Span (R) 65) (these are, for example, Sigma (St Louis) s, MO)). Sorbitan sesquioleate (Span® 83) is available from Aldrich Chemical Co. , Inc. (Milwaukee, WI). Other surfactants that can be used include polyoxyethylene sorbitan (Tween®) compounds. Exemplary cosurfactants include polyoxyethylene sorbitan monolaurate (Tween® 20 and Tween® 21), polyoxyethylene sorbitan monooleate (Tween® 80). And Tween® 80R), polyoxyethylene sorbitan monopalmitate (Tween® 40), polyoxyethylene sorbitan monostearate (Tween® 60 and Tween® 61), polyoxy Ethylene sorbitan trioleate (Tween® 85) and polyoxyethylene sorbitan tristearate (Tween® 65) (for example, Sigma (St Louis, MO)) Available). Other exemplary commercially available surfactants include polyethyleneoxy (40) -sorbitol hexaoleate ester (Atlas G-1086, ICI Specialties, Wilmington DE), hexadecyltrimethylammonium bromide (CTAB, Aldrich), and direct Examples include chain alkylbenzene sulfonate (LAS, Ashland Chemical Co., Columbia, OH).

  Other exemplary surfactants include fatty acid soaps, alkyl and dialkyl phosphates, alkyl sulfates, alkyl sulfonates, primary amine salts, secondary amine salts, tertiary amine salts, quaternary amine salts, n-alkyls. Xanthate, n-alkyl ethoxylated sulfate, dialkyl sulfosuccinate salt, n-alkyl dimethyl betaine, n-alkyl phenyl polyoxyethylene ether, n-alkyl polyoxyethylene ether, sorbitan ester, polyethylene oxysorbitan ester, sorbitol ester And polyethyleneoxysorbitol esters.

  Other surfactants include lipids (eg, phospholipids, glycolipids, cholesterol and cholesterol derivatives). Exemplary lipids include fatty acids or molecules containing fatty acids (wherein the fatty acids include, for example, palmitate, oleate, laurate, myristate, stearate, arachidate, behenate, lignocerate, palmitate, linoleate, linolenate). And arachidonates), and salts thereof (e.g., sodium salts). Fatty acids can be modified, for example, by converting acid functional groups to sulfonates by coupling reactions to small molecules containing acid functional groups, or by other functional group transformations known to those skilled in the art.

  Furthermore, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), starch and their derivatives may find use as surfactants in the present invention.

  Cationic lipids can be used as cosurfactants (eg, cetyltrimethylammonium bromide / chloride (CTAB / CTAC), dioctadecyldimethylammonium bromide / chloride (DODAB / DODAC), 1,2-diacyl-3-trimethyl. Ammonium propane (DOTAP), 1,2-diacyl-3-dimethylammonium propane (DODAP), [2,3-bis (oleoyl) propyl] trimethylammonium chloride (DOTMA), and [N- (N′-dimethylamino) Ethane) -carbamoyl] cholesterol, dioleoyl) (DC-Chol)). Alcohols can also be used as cosurfactants (eg, propanol, butanol, pentanol, hexanol, heptanol and octanol). Other alcohols with longer carbon chains can also be used.

  Incorporation of drug molecules into an article: The terms “drug”, “drug conjugate”, “bioactive agent” and “therapeutic agent” are used interchangeably herein. In a preferred embodiment, the drug molecule is covalently bound to the article scaffold. For example, the chemotherapeutic agent doxorubicin is a combination of the amine moiety on doxorubicin and the carboxylic acid moiety contained in the hydrogel scaffold by using, for example, sodium acrylate (NaA), malonic acid acrylamide (MalAc) or CiBA as building blocks. It can be coupled to the scaffold via an EDC coupling reaction between. In another embodiment, doxorubicin can be linked via an imine bond by reacting the amine moiety of doxorubicin with the aldehyde moiety of the hydrogel scaffold. Aldehydes can be made by first forming an article using a carbohydrate-based building block and then oxidizing the carbohydrate after the article is formed. In another embodiment, doxorubicin can be attached to the article matrix via its ketone moiety. Carbohydrazide or other dihydrazide or di-amino-oxy functionalized structures can be used to attach doxorubicin to scaffolds containing aldehydes or ketones through the formation of hydrazone bonds. Aldehydes or ketones can be incorporated into the scaffold through the use of ketone-containing acrylate building blocks (eg, DAA). Hydrazone binding can conveniently release therapeutic compounds under mildly acidic physiological conditions that encounter article endocytosis and entry into lysosomes.

  In another embodiment, by including an APMA or methacrylate functionalized short peptide (prepared according to US Pat. No. 5,037,883) building block, for example, a nanoarticle scaffold composed of amino groups is It can be used to covalently attach cyclosporine containing carboxylate linkages. Cyclosporine drugs may find pathologies that benefit from immunosuppression (eg, inflammatory diseases) and applications for organ transplantation.

  In another embodiment, a nanoarticle scaffold composed of aldehyde or ketone groups (eg, incorporated by including DAA, levulinic acid acrylamide or oxidized carboxylate (eg, inulin or dextran building blocks)) is a hydrazone coupling. Through the use of the scheme, it can be used to covalently link drugs or drug derivatives (eg, calicheamicin) containing certain moieties. This coupling scheme results in a low pH hydrolytically unstable hydrazone bond found especially in lysosomes (Bernstein I. et al., Bioconjugate Chem., 2002, 13, 40-46).

  In another embodiment, a nanoarticle scaffold composed of acid or anhydride groups, incorporated, for example, by including sodium acrylate or anhydride building blocks, shares dexamethasone via the use of an amide coupling scheme. Can be used to combine reservoirs.

  Nanoarticle scaffolds composed of carboxylate groups (eg, incorporated by including sodium acrylate (NaA), CiBA, or MAIAc building blocks) are amine moieties, drugs or drug derivatives (eg, peptide modified camptothecin (Frigerio E , Et al., J. Controlled Release, 2000, 65, 105-119)) can be used to covalently link via an EDC-NHS coupling scheme.

  In another embodiment, when the nanoarticle scaffold is composed of aldehyde or ketone groups, which can be incorporated through the use of DAA, levulinic acid acrylamide or oxidized carboxylate (eg, inulin or dextran) Drugs or drug derivatives bearing amines (eg, gemcitabine) can be incorporated through the use of a “Schiff base” coupling scheme. The imide bond formed from the binding of gemcitabine to DAA can be cleaved by acidic media. During internalization, the drug is taken up by cells exposed to the acidic environment of lysosomes, thereby releasing gemcitabine in its unmodified form.

  In another embodiment, a nano-article scaffold consisting of a carbonyl group (eg, incorporated through the inclusion of a CiBA, MalAc or NaA building block) can be linked via the use of an EDC-NHS coupling scheme. For example, it can be used to covalently bond to drugs and drug derivatives including salicylic acid). For example, the hydroxyl group of salicylic acid reacts with the carboxyl group of CiBA, MalAc or NaA to form an ester bond. Hydrolysis or esterase enzymes cleave the ester bond between salicylic acid and the carboxyl group of CiBA or NaA, releasing the unmodified form of salicylic acid.

  In another embodiment, the drug structure can be modified to facilitate binding of the nanoarticle to the scaffold. For example, the 2 'hydroxyl group of paclitaxel can be reacted with multiple linkers that can be coupled to the scaffold of the nanoarticle. For example, the acidic moiety of a resin-immobilized glycine linker can be coupled to paclitaxel using carbodiimide; the resulting compound can be cleaved at the amine site using 1% TFA, using an EDC coupling scheme. Free amines can be produced that can be conjugated with nano-items having carboxylic acids.

  In another embodiment, attachment of paclitaxel-2′-succinate (Deutsch H. et al., J Med. Chem., 1989, 32, 788-792) to a nanoarticle uses carbodiimide-mediated amide coupling. Is possible. This coupling occurs between the paclitaxel-2'-succinate group and the amine group of the APMA component of the nanoarticle to form an unstable ester.

  In another embodiment, the nanoarticle can be directly attached to paclitaxel by reacting an acid functionalized (NaA) nanoarticle with the 2'-hydroxyl group of paclitaxel. This chemical pathway has been previously described using a poly (L-glutamic acid) -paclitaxel conjugate (Li H. et al., Cancer Res., 1998, 58, 2404-2409).

  In another embodiment, a nanoarticle scaffold comprising a carboxylic acid (e.g., incorporated via encapsulation of sodium acrylate (NaA) building blocks) comprises an amine functionalized 5FU derivative and a carboxylic acid located on the nanoarticle. Can be used to covalently link drugs and derivatives of drugs containing moieties (eg, 5-fluorouracil (5FU) (or derivatives thereof)) through the use of amine-forming coupling reactions with acids . The synthesis of 1-alkylcarbonyloxymethyl derivatives of 5FU has been described previously and these materials have been demonstrated to release unmodified forms of 5FU (Taylor HE; Sloan KB). , Journal of Pharmaceutical Sciences, 1998, 87, 15). Application of this synthetic route necessarily results in an amine functionalized 5FU while achieving a similar release profile.

  Nanoarticle scaffolds composed of acid groups (eg, incorporated through the inclusion of CiBA, MalAc or NaA building blocks) are covalently attached to drugs or drug derivatives containing carboxylic acid moieties (eg, methotrexate) Can be used for. This covalent linkage is accomplished by first coupling the drug or derivative of the drug to boc-protected ethanolamine to form an ester, then deprotecting the modified drug and then via the EDC coupling scheme To be coupled. This ester conjugate is known to hydrolyze at low pH to release the original form of the drug (Wilson JM et al., Biochem Biophys. Res, Commun., 1992, 184, 300-305; Ohkuma S., Poole B., Proc. Natl. Acad. Sci. USA, 1978, 75, 3327-3331). Such low pH conditions are found in intracellular lysosomes. These nanoarticles may find use in the treatment of multiple pathologies, including cancer and inflammatory conditions such as rheumatoid arthritis and inflammatory bowel disease.

  In another embodiment, a platinum conjugate is incorporated on the nanoarticle scaffold. The platinum may be in the second stage or fourth stage oxidation state. There remains a clear need for new platinum chelates that are further improved in therapeutic indications compared to currently approved platinum chelates. Such chelates are highly water-soluble and stable in aqueous environments, but are sufficient in tumor cells to provide species that can cross-link DNA and ultimately cause tumor cell death. Should be unstable. The hydrogel network of nano-articles of the present invention, combined with the ability to fine-tune its chemical composition, allows a high degree of water solubility and flexibility, such as adhering to chelates. Constitutes a significant improvement over WO 9847537. It has been shown that changes in the structure of platinum chelates can alter the type of tumor type for which platinum treatment is effective and / or change the toxicity profile of the chelate. In one important embodiment of the invention, platinum is expected to complex with the hydrogel matrix via O, N-bonds, resulting in a more stable compound.

  This includes nano-articles obtained by free radical polymerization, and acid functional groups (eg, derived from NaA) and amine moieties (eg, derived from APMA) or amide moieties (eg, acrylamide), as well as both types Nanoarticles comprising a combination of building blocks that retain functional groups (CIBA, MalAc or methacrylolate functionalized short peptides produced according to US Pat. No. 5,037,883) can be preferably achieved. Such moieties provide a point of attachment for producing O, N-cis platinum nanoarticle conjugates and open the possibility of targeting nanoarticles.

  Finally, the high toxicity of platinum derivatives makes specific targeting of nanoarticles to organs or tumor sites wonderfully effective for increasing therapeutic indices.

  Drugs that may find use in the present invention include peripheral nerves, adrenergic receptors, cholinergic receptors, nervous system, skeletal muscle, cardiovascular system, smooth muscle, blood circulation system, synaptic site, neuro-effector Junction site, endocrine system, hormone system, immune system, reproductive system, skeletal system, otacoid system, digestive system and excretory system, histamine system, respiratory system, reticuloendothelial system, skeletal system, skeletal muscle, smooth muscle, immune system Drugs that act in the reproductive system, cancerous tissue, and the like. Active drugs that can be delivered to act in these recipients include anticonvulsants, analgesics, antiparkinson drugs, anti-inflammatory drugs, calcium antagonists, anesthetics, antimicrobials, antimalarials, antiparasitics , Antihypertensive, antihistamine, antipyretic, alpha-adrenergic agonist, alpha blocker, biocide, bactericidal, bronchodilator, beta-adrenergic blocker, contraceptive, chemotherapeutic, cardiovascular system Drugs, calcium channel inhibitors, suppressors, diagnostics, diuretics, electrolytes, enzymes, hypnotics, hormones, hypoglycemic drugs, hyperglycemic drugs, muscle contractors, muscle relaxants, neoplastic drugs, glycoproteins, nucleoproteins , Lipoproteins, ophthalmics, physic energizers, sedatives, steroids, sympathomimetic drugs, side effects Neuroid agents, analgesics, urinary tract drugs, vaccines, 窒用 drugs, vitamins, nonsteroidal anti-inflammatory drugs, angiotensin converting enzymes, polynucleotides, polypeptides, and the like polysaccharides include, but are not limited to.

  In presently preferred embodiments, drugs that can be advantageously used in the present invention include chemotherapeutic agents (e.g., doxorubicin, paclitaxel, gemcitabine, vincristine, cisplatin, carboplatin, chlorambucil, topotecan, methotrexate, of these compounds. Derivatives, and other FDA-approved chemotherapeutic drugs), as well as molecules that are not yet commercially available but can act as chemotherapeutic drugs, and derivatives and analogs of all the above chemotherapeutic drugs, It is not limited.

  The therapeutic agents for delivery of the present invention can be in a variety of pharmaceutically acceptable forms (eg, prodrugs, uncharged molecules, molecular complexes, and pharmaceutically acceptable salts). Pharmaceutical derivatives such as esters, ethers and amides can be used.

  Functionalization of the article with recognition elements: After the assembled building block is cross-linked to form a hydrogel scaffold and the therapeutic agent is covalently attached to the scaffold, the article surface can be functionalized with RE. . The RE can be attached to the surface of the nanoarticle directly or through a linker molecule. In the linker form, some or all of the RE is “presented” at the end of this tether. To that end, in one application of the invention, the article consists of RE presented on a hydrogel scaffold. In another embodiment of this patent, the article consists of an RE (eg, a high affinity peptide) attached to the surface of the core scaffold of the article via a linker molecule, which preferably is a polyethylene glycol ( PEG).

  For each of these embodiments, the article can be functionalized with several coupling strategies, both the various components and the order of addition of the reactive chemical moieties used in the coupling. fluctuate.

  The components can be combined with each other in the following order. The hydrogel scaffold is first reacted with a tether containing a bifunctional PEG, followed by functionalizing some free ends of the PEG chain with RE. Alternatively, RE is first coupled to a PRG containing tether, followed by coupling of the other PEG terminus to the scaffold.

  Several combinations of reactive moieties can be selected to bind the RE to the tether and to react the tether with the scaffold. When using a series of orthologous reaction sets, when changing some of the building blocks of the scaffold, and / or when anchoring the arms, REs can be placed on different scaffolds on the same article scaffold in a well-controlled ratio. It is also possible to bind to different molecular structures that bind. Reactions using orthogonal reactive pairs can be performed simultaneously or sequentially.

  As far as the reaction conditions are concerned, it is preferred to functionalize the article in an aqueous system. Surfactant and oil phases, hydrogel scaffold synthesis residue, use of solvent wash (single or in combination) (eg, solubilize surfactant and oil while article is settling) Can be removed through the use of ethanol to use; the use of surfactant-absorbing beads; the use of dialysis; or the use of an aqueous system (eg, 4M urea). Methods for removing the surfactant are known in the art.

  The RE must contain functional groups that allow attachment to the article. Preferably, but not necessarily, this functional group is one member of a pair of chemoselective reagents selected to assist in the coupling reaction (Lemieux, G., Bertozzi, C., Trends in Biotechnology, 1998, 16, 506-513). For example, when the article surface (and / or a linker grafted to the surface) presents a haloacetal, the peptide RE can be attached via a sulfhydryl moiety. Sulfhydryl moieties in the RE structure can be achieved through the inclusion of cysteine residues.

  Coupling is also possible between the primary amine on the article or linker end and the carboxylic acid on the RE. Carboxylic acids in the peptide structure can be found either on the amino terminus (for linear peptides) or through the inclusion of aspartic acid or glutamic acid residues. The opposite form in which the carboxylic acid is present on the primary amine belonging to the article and peptide is also easily accessible. Many polymerizable building blocks contain acidic moieties that are accessible on the surface of the beads after polymerization. Like poly (amino acid) based REs, primary amine functionality can be found either through the N-terminus (if linear) and / or through the introduction of lysine residues.

  Another example of a reactive chemical pair consists of the coupling of a sulfhydryl with a haloacetal or maleimide moiety. Maleimide functional groups can be synthesized by other common functional groups (described by way of example by GT Hermanson (Bioconjugate Technologies, Academic Press, 1996)). For example, by reacting a carboxylic acid, amine, thiol or alcohol) with a linker, it can be easily introduced either on the peptide, on the linker, or on the surface of the article. In preferred embodiments, encapsulation of CiBA, or other disulfide-containing building blocks, in a scaffold facilitates binding of RE via a thiol reactive moiety. After scaffold formation, reduction of CiBA disulfide bonds yields free thiols. A linker molecule containing a thiol-reactive group (eg, bromoacetamide or maleimide) is added to the article containing the reduced therapeutic agent to attach the linker to the article scaffold. The RE is then added and reacts with the free end of the linker moiety, resulting in an RE functionalized article. Alternatively, the RE can be attached to one end of the linker molecule prior to attaching the linker molecule to the reducing article.

  The peptide can also be attached to the article and / or tether by reaction between the aminooxy functionality and the aldehyde or ketone moiety. The aminooxy moiety (either on the article or on the peptide) can be introduced by a series of variations known in the art starting from other common functional groups (eg amines). Similarly, articles containing aldehydes or ketones and aldehyde-containing peptides can be readily synthesized by known methods.

  The resulting RE functionalized drug-containing article can be used immediately, stored as a liquid solution, or lyophilized for long-term storage.

  The RE can be a small or high molecular structure that provides the desired binding interaction with the cell surface receptor of the target molecule. The number of recognition element portions per article can range from 2 to 1000, preferably from 2 to 500, and most preferably from 2 to 100. An article can be composed of more than one type of RE, if desired. As used herein, a “type” of RE is defined as a specific molecular structure.

The RE preferably consists of a peptide. In accordance with the present invention, peptides used as RE generally have a dissociation constant between 10 ⁻⁴ and 10 ⁻⁹ M or less. Such RE can consist of known peptide ligands. For example, the Phoenix Peptides peptide ligand-receptor library (http://www.phoenixpeptide.com/Peptidelibrarylist.htm) contains thousands of known peptide ligands for receptors of potential therapeutic value. The peptides can be natural peptides such as lactams, dalargin and other encaphalins, endorphins, angiotensin II, gonadotropin releasing hormone, melanocyte stimulating hormone, thrombin receptor fragment, myelin, and antigenic peptides. Peptide binding blocks useful in the present invention are discovered through high-throughput screening of peptide libraries (eg, phage display libraries or libraries of linear sequences displayed on beads) against the protein of interest. obtain. Such screening methods are known in the art (eg, C.F. Barbas, D.R. Burton, JK. Scott, G.J. Silverman, Page Display, 2001, Cold Spring Harbor Laboratory Press. , Cold Spring Harbor, NY). High affinity peptides can consist of naturally occurring amino acids, modified amino acids or fully synthetic amino acids. The length of the recognition portion of the peptide can vary from about 3 to about 100 amino acids. Preferably, the recognition portion of the peptide ranges from about 3 to about 15 amino acids, and more preferably from 3 to 10 amino acids. Shorter sequences are preferred. This is because peptides with less than 15 amino acids may be less immunogenic than longer peptide sequences. Small peptides have the additional advantage that the library can be easily screened. Alternatively, they can be more easily synthesized using solid phase techniques.

  The RE can include a variety of other molecular structures, including antibodies, antibody fragments, lectins, nucleic acids, and other receptor ligands. Humanized antibodies or fully human antibodies, as well as humanized antibody fragments or fully human antibody fragments are preferred for use in the present invention.

  Furthermore, it may be possible to design other non-protein compounds to be used as binding moieties using techniques known to those skilled in the art of drug design. Such methods include, but are not limited to, coherent field (SCF) analysis, configuration interaction (CI) analysis, and normal mode dynamics computer programs, all of which are detailed in the scientific literature. It is described in. See Rein et al., Computer-Assisted Modeling of Receptor-Ligand Interactions, Alan Liss, New York (1989). The preparation of non-protein compounds and moieties can depend on their structure and other features, and can usually be accomplished by standard chemical synthesis techniques. For example, Methods in Carbohydrate Chemistry, Vols. I-VII; Analysis and Preparation of Sugars, edited by Whistler et al., Academic Press, Inc. , Orlando (1962), the disclosure of which is incorporated herein by reference.

By using multiple RE molecules of the same molecular structure or multiple RE molecules of different molecular structure to make up the article, the binding force of the article can be increased. As used in the present invention, “high affinity” means that a single RE binds to a single target molecule with a binding constant stronger than 10 ⁻⁴ M, while “Avidity” means that two or more such RE units bind to two or more target molecules on a cell or on a molecular complex.

  These REs can target multiple disease-related biomolecules. As a tumor-related target, erbB1 (for example using growth factor EGF or a peptide consisting of the amino acid sequence YCPIWKFPDECY, or Greene, et al., J. Biol. Chem., 2002, 277 (31), 28330-28339 ErbB2 (for example, peptides composed of the amino acid sequence CdFCDGFdYACYMDV (where dF and dY represent the D isomers of these amino acid residues) or Murali, J. et al. Chem., Chem., 2001, 44, 2565-2574 using other sequences depicted as RE), erbB3, erbB4, CMET, CEA (eg other disclosed as RE in PCT WO 01/74849) peptide As well as EphA2. Amino acid sequences as described in VEGFR-1, VEGFR-2 (e.g., Demangel et al., EMBO J., 2000, 19 (7), 155-1533) as targets for blood vessels associated with multiple pathologies, including cancer. Extracellular peptides such as integrins (including integrin αvβ3 and integrin αvβ1), and fibrin (which uses peptides composed of ATWLPPR), which are derived from the amino acid sequence disclosed in PCT publication WO 02/055544. Can be targeted using

(Item characteristic value :)
As implemented in the present invention, the article has several characteristic values that make them excellent therapeutic candidates.

  The article can be administered by injection (subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, intracerebral, or parenteral), with intravenous injection being the preferred route. The article may be suitable for nasal delivery, pulmonary delivery, vaginal delivery, ocular delivery and oral administration. The article can be suspended in a pharmaceutically acceptable carrier for administration.

  Reagents and starting materials in some embodiments are Sigma-Aldrich (St Louise, MO and Milwaukee, Wis.), Kodak (Rochester, NY), Fisher (Pittsburgh, PA), Pierce Chemical Company (IL), Pierce Chemical Company (IL). Inc. (Westborough, MA), Radcure (Smyrna, GA), and chemical wholesalers such as Polysciences (Niles, IL). PEG compounds can be purchased through NOF America Corporation (White Plains, NY) and Nektar (Birmingham, AL). Peptides used as REs can be purchased from a number of sources, one from Bachem (King of Prussia, PA). Proteins can be obtained from sources such as Calbiochem (San Diego, Calif.).

  The following non-limiting examples are provided to further describe how the invention can be practiced.

(Example 1: Synthesis of IMMA (inulin multimethacrylate))
Inulin (4 g) was weighed into a single neck round flask. A Teflon-coated stir bar was added and the flask was sealed with a septum. Anhydrous pyridine (approximately 20 mL for each 5 grams of inulin) was then transferred into the flask, keeping the entire system under a nitrogen atmosphere. The mixture was stirred until the inulin was dissolved, then 1 mL of methacrylic anhydride was added dropwise using a syringe. The reaction was continued for 16 hours. After this period, pyridine was sufficiently removed under reduced pressure to obtain a viscous liquid. Toluene (about 40 mL) was then added with vigorous mixing to precipitate the crude product. The liquid was then decanted from the precipitate. This solid was dissolved in water to produce a viscous but free flowing syrup that was then precipitated with 2-propanol (about 200 mL). This water dissolution and subsequent precipitation process was repeated more than once, after which the product was dried under reduced pressure. The product was then redissolved in 50 mL of water and filtered through filter paper to remove accumulated debris and other insolubles, and the product was then lyophilized to yield inulin multimethacrylate (IMMA). The identity of IMMA (the degree of methacrylate functionalization) was confirmed by NMR analysis.

(Example 2: Preparation of dextran double (methacrylate) (DMMA))
Dextran (molecular weight = 5000) was dissolved in 100 mL dry DMSO with the aid of a stir bar. Upon complete dissolution, anhydrous pyridine was added (20 mL). Throughout the 0.5 hour step, methacrylic anhydride (3.27 mL) was added dropwise and the reaction was allowed to stir overnight. The next day, the product was precipitated by the addition of toluene (150 mL) and the reaction solvent / toluene mixture was decanted from the solid. This product was dissolved in the minimum amount of water needed to obtain a syrup and added to rapidly stirred isopropanol (ca. 150 mL). The solution was decanted from the solid and the dissolution / precipitation cycle was repeated two more times. The solid was dried under reduced pressure to remove a trace amount of organic solvent, dissolved in 75 mL of water, and the resulting solution was filtered. The product was isolated by lyophilization and characterized by ¹ H NMR.

(Example 3: Synthesis of CiBA)
Sodium hydroxide (2.0 g) was dissolved in 70 mL of dry methanol. L-cystine (2.73 g) was added to the methanolic NaOH solution and the round bottom flask containing the mixture was immersed in an ice water bath to maintain the reaction vessel at 0 ° C. Acryloyl chloride (2.22 mL) was then added dropwise to the methanolic cystine solution.

The reaction was capped and stirred for 1 hour at room temperature, after which the reaction solution was centrifuged and the liquid phase decanted into stirred ethyl acetate (120 mL). The resulting suspended solid was isolated by centrifugation and dried under reduced pressure. The identity of this isolated material as N, N′-cystine bisacrylamide (CiBA) was confirmed by ¹ H NMR.

(Example 4: Synthesis of MalAc (malonate-acrylamide))
Diethylaminomalonate hydrochloride (5 g) is weighed into a single neck round flask. A Teflon-coated stir bar is added and the flask is sealed with a septum. Anhydrous dichloromethane (approximately 10 mL for each gram of diethylaminomalonate) is then transferred into the flask, keeping the entire system under a nitrogen atmosphere. The mixture is stirred and 3.62 mL of triethylamine is added via syringe (1.1 equiv) followed by 2.12 mL of acryloyl chloride (1.1 equiv) added dropwise via syringe. The reaction was continued for 2 hours. The reaction mixture is extracted three times with water to remove unreacted organisms [triethylamine and acrylic acid]. The organic phase is dried under reduced pressure and resuspended in water containing a resin having a strongly acidic residue. The aqueous resin suspension is placed on a rotary shaker and the deprotection reaction is allowed to proceed overnight. The reaction mixture is then filtered and lyophilized to obtain malonic acid acrylamide.

(Example 5: Synthesis of PEG-1500 dBA)
Poly (ethylene glycol) (average molecular weight = 1500, 15.36 g) was dissolved in 75 mL of dry chloroform contained in a round bottom flask. A stir bar was added to assist in the dissolution process and to maintain the homogeneity of the reactants. Bromoacetyl chloride (4.00 mL) was added, an air-cooled reflux condenser was placed in contact with the round bottom flask, and the reaction mixture was heated at reflux under a nitrogen purge vented to atmosphere (HCl gas generated during the reaction). To remove). After 4 hours, more bromoacetyl chloride (1.0 mL) was added and the reaction mixture was heated for an additional 5 hours. The reaction mixture was cooled and gently stirred overnight. The next day, the solvent and excess reagent were removed under reduced pressure and the residue was dissolved in saturated sodium bicarbonate. The aqueous solution was extracted with chloroform (4 times, 50 mL). The organic extracts were combined, dried under magnesium sulfate and filtered. Removal of this solvent under reduced pressure leaves PEG-1500 dBA as an off-white solid. The identity of this product was confirmed by ¹ H NMR.

Example 6: Preparation of heterobifunctionalized PEG-400, bromoacetate and carboxylic acid
Poly (ethylene glycol) dibromoacetate (average molecular weight 400, PEG-400 dBA) (4.0 g) was dissolved in phosphate buffer (0.15 M) (150 mL, pH = 8) containing 100 mL of THF. To this solution was added 3-mercaptopropionic acid (0.17 g) in 10 mL of water by rapid mixing provided by a stir bar. The pH was adjusted again to 8 by the addition of 1.0 M NaOH solution. Sixteen hours after the addition, the reaction volume was reduced to 50 mL under reduced pressure. The volume is increased to 150 mL by adding 100 mL, pH = 8.0 phosphate buffer (0.10 M), and the aqueous solution is extracted with chloroform (2 × 50 mL) to remove unreacted PEG starting material. Removed. The pH of the solution was adjusted to 2 by adding 1.0 M HCl and the solution was extracted again with chloroform (3 times 50 mL). The combined extracts of this pH = 2.0 solution were dried over sodium sulfate and filtered. Removal of the solvent under reduced pressure gave the target compound.

Example 7: Preparation of heterobifunctionalized PEG-200, thiol and carboxylic acid
Poly (ethylene glycol) dithiol (average molecular weight 200) (1.53 g) was dissolved in 100 mL of THF-containing phosphate buffer (0.15 M) (150 mL, pH = 8). To this solution was added bromoacetic acid (0.27 g) in 10 mL of water by rapid mixing provided by a stir bar. The pH was adjusted again to 8 by the addition of 1.0 M NaOH solution. After 16 hours of this addition, the solvent was removed under reduced pressure to give a viscous residue to which 100 mL of phosphate buffer (pH = 8.0) was added (0.050M). The aqueous solution was extracted with chloroform (2 times, 50 mL) to remove unreacted PEG starting material. The pH of the solution was adjusted to 2 by adding 1.0 M HCl and the solution was extracted again with chloroform (3 times 50 mL). The combined extracts of this pH = 2.0 solution were dried over sodium sulfate and filtered. Removal of the solvent under reduced pressure gave the target compound.

(Example 8: Formation of IMMA-CiBA-NaA skeleton through free radical polymerization)
The aqueous phase was 83 wt% buffer, 14 wt% IMMA, 2 wt% CiBA, 1 wt% sodium acrylate, and eosin Y (this photoinitiator is 0.001 to 0.1 wt% of monomer mass Present). An oil + surfactant phase was prepared by mixing 7.3 wt% Igepal CO-210, 9.4 wt% Igepal CO-720, and 83.3% wt cyclohexane. 3 grams (3 g) of this aqueous phase was added to 30 g of this oil + surfactant phase with vigorous stirring, causing the formation of a reverse microemulsion. This inverse microemulsion comprises surfactant stabilized aqueous phase nanodroplets dispersed in a continuous phase of cyclohexane. The inverse microemulsion was transferred to a 100 mL Schlenk tube and degassed by briefly vacuuming in this cooled mixture. The contents of the Schlenk tube were stirred and irradiated with a UV light source for 1 hour to polymerize the basic elements. Once the polymerization was complete, the nanoarticle was precipitated by adding pure ethanol directly to the solution and isolated from the reaction mixture by centrifugation. The nanoarticle-containing pellet was resuspended in deionized water. Residual surfactant and solvent were removed by standard techniques (solid phase extraction). At this point, the aqueous solution of nanoarticle was filtered and the product was isolated as a solid product after lyophilization.

(Example 9: Generation of IMMA-CiBA-DAA skeleton through free radical polymerization)
The aqueous phase was prepared by combining 83 wt% water, 14 wt% IMMA, 2 wt% CiBA and 1 wt% diacetone acrylamide (DAA). The oil + surfactant phase was prepared by mixing Igepal CO-210, Igepal CO-720 and cyclohexane in a weight ratio of 1.0: 1.3: 9.0. 3 grams (3 g) of this aqueous phase was mixed with 30 g of this oil + surfactant phase, causing the formation of a reverse microemulsion. This inverse microemulsion contained surfactant-stabilized aqueous phase nanodroplets dispersed in a continuous phase of cyclohexane. To this inverse microemulsion was added eosin Y-containing aqueous solution (this photoinitiator represents 0.001 to 0.1% by weight of the monomer mass). The microemulsion was degassed by a freeze-thaw cycle under reduced pressure (with backfilling of N ₂ gas between cycles). The contents were agitated and polymerized by irradiating with a UV light source or visible light source of at least 100 W for 20 minutes to 2 hours. Once the polymerization was complete, the nanoarticle was precipitated by adding 9 mL of pure ethanol directly to the solution. The nanoarticle-containing pellet was resuspended in water. Residual surfactant and solvent were removed by standard techniques (dialysis, chromatography, etc.). At this point, this aqueous solution of nanoarticles can be lyophilized if desired.

(Example 10: IMMA-CiBA-APMA scaffold formation by free radical polymerization)
The aqueous phase was mixed with 83 wt% buffer, 14 wt% IMMA, 2 wt% CiBA, 1 wt% N- (3-aminopropyl) methacrylamide hydrochloride (APMA), and eosin Y (the photoinitiator was .0. (Representing a monomer mass of 001 to 0.1% by weight). The oil + surfactant phase was prepared by mixing 7.3 wt% Igepal CO-210, 9.4 wt% Igepal CO-720, and 83.3% wt cyclohexane. 3 grams (3 g) of the aqueous phase was added to 30 g of oil + surfactant phase with vigorous stirring, resulting in the formation of a reverse microemulsion. The inverse microemulsion comprises aqueous phase surfactant-stabilized nanodroplets dispersed in a continuous phase of cyclohexane. The inverse microemulsion was transferred to a 100 mL Schlenk tube and degassed by briefly pulling a vacuum with the chilled mixture. The contents of the Schlenk tube were agitated and irradiated with a UV light source for 1 hour to polymerize the building blocks. Once the polymerization was complete, the nanoarticles were precipitated by adding pure ethanol directly to the solution and isolated from the reaction mixture by centrifugation. The pellet containing the nanoarticle was resuspended with deionized water. The remaining surfactant and solvent were removed by standard techniques (solid phase extraction). At this point, the aqueous solution of nanoarticles was filtered and the product was isolated as a solid product after lyophilization.

(Example 11: IMMA-MalAc scaffold formation by free radical polymerization)
Aqueous phase with 80 wt% buffer, 14 wt% IMMA, 6 wt% malonic acrylamide (MalAc), and eosin Y (photoinitiator represents 0.001 to 0.1 wt% monomer mass). Prepared by combining. The oil + surfactant phase was prepared by mixing 7.3 wt% Igepal CO-210, 9.4 wt% Igepal CO-720, and 83.3% wt cyclohexane. 3 grams (3 g) of the aqueous phase was added to 30 g of oil + surfactant phase with vigorous stirring, resulting in the formation of a reverse microemulsion. The inverse microemulsion comprises aqueous phase surfactant-stabilized nanodroplets dispersed in a continuous phase of cyclohexane. The inverse microemulsion was transferred to a 100 mL Schlenk tube and degassed by briefly pulling a vacuum with the chilled mixture. The contents of the Schlenk tube were agitated and irradiated with a UV light source for 1 hour to polymerize the building blocks. Once the polymerization was complete, the nanoarticles were precipitated by adding pure ethanol directly to the solution and isolated from the reaction mixture by centrifugation. The pellet containing the nanoarticle was resuspended with deionized water. The remaining surfactant and solvent were removed by standard techniques (solid phase extraction). At this point, the aqueous solution of nanoarticles was filtered and the product was isolated as a lyophilized powder.

(Example 12: Scaffold containing short N-methacryloylated peptide)
The aqueous phase was composed of 82 wt% buffer, 14 wt% IMMA, 2 wt% CIBA and 2 wt% N-methacryloylated Gly-Gly peptide (made according to US Pat. No. 5,037,883) and eosin Y ( The photoinitiator was prepared by combining 0.001-0.1 wt% monomer mass). The oil + surfactant phase was prepared by mixing 7.3 wt% Igepal CO-210, 9.4 wt% Igepal CO-720, and 83.3% wt cyclohexane. 3 grams (3 g) of the aqueous phase was added to 30 g of oil + surfactant phase with vigorous stirring, resulting in the formation of a reverse microemulsion. The inverse microemulsion comprises aqueous phase surfactant-stabilized nanodroplets dispersed in a continuous phase of cyclohexane. The inverse microemulsion was transferred to a 100 mL Schlenk tube and degassed by briefly pulling a vacuum with the chilled mixture. The contents of the Schlenk tube were agitated and irradiated with a UV light source for 1 hour to polymerize the building blocks. Once the polymerization was complete, the nanoarticles were precipitated by adding pure ethanol directly to the solution and isolated from the reaction mixture by centrifugation. The pellet containing the nanoarticle was resuspended with deionized water. The remaining surfactant and solvent were removed by standard techniques (solid phase extraction). At this point, the aqueous solution of nanoarticles was filtered and the product was isolated as a lyophilized powder.

(Example 13: Scaffold formation by reaction of oxidized inulin and bisaminooxy compound)
The aqueous phase was prepared by mixing with 85 wt% water and 15 wt% inulin. The oil + surfactant phase was prepared by mixing Igepal CO-210, Igepal CO-720 and cyclohexane in a weight ratio of 1.0: 1.3: 9.0. 3 grams (3 g) of the aqueous phase was mixed with 40 g of oil + surfactant phase resulting in the formation of an inverse microemulsion. 1 g of aqueous sodium periodate was added to the mixture. In situ oxidation was allowed to proceed for about 10 minutes. A concentrated solution (at least 1 g / ml) in an aqueous methanol mixture (50/50 vol) of bis [(2-amino-oxy) ethylamide]-(1,3-) propane was then added to the microemulsion and Reacted overnight. The resulting nanoarticles obtained by cross-linking of bis [(2-amino-oxy) ethylamide]-(1,3) propane using the aldehyde functionality of oxidized inulin are precipitated by precipitation and centrifugation in the presence of ethanol. Isolated and purified from excess unreacted reagent by dialysis.

(Example 14: Scaffold formation by reaction of oxidized dextran and carbohydrazide)
The aqueous phase was prepared by mixing with 90 wt% water and 10 wt% dextran. The oil + surfactant phase was prepared by mixing Igepal CO-210, Igepal CO-720 and cyclohexane in a weight ratio of 1.0: 1.3: 9.0. 3 grams (3 g) of the aqueous phase was mixed with 40 g of oil + surfactant phase resulting in the formation of an inverse microemulsion. 1 g of sodium periodate aqueous solution was added to the mixture so that there was a maximum of 1 equivalent periodate per glucose monomer unit (from dextran). In situ oxidation was allowed to proceed for 5-20 minutes. A concentrated solution (at least 1 g / ml) of carbohydrazide (50-250 mM, pH 8-9) in buffer solution was then added to the microemulsion and allowed to react overnight at 4 ° C. The resulting nanoarticles obtained by cross-linking of carbohydrazide using the aldehyde functional group of oxidized dextran were isolated by precipitation in the presence of ethanol and centrifugation and purified from excess unreacted reagent by dialysis.

(Example 15: Scaffold formation by reaction of oxidized inulin and bishydrazineoxy compound)
The aqueous phase was prepared by mixing with 85 wt% water and 15 wt% inulin. Two grams (2 g) of this aqueous phase was mixed with 30 g of oil + surfactant phase composed of 8.5 wt% Igepal CO-520 in cyclohexane. Periodic acid solution in 1 g of water was added to the mixture so that there was a maximum of 1 equivalent periodate per glucose monomer unit (derived from inulin). In situ oxidation was allowed to proceed for about 10 minutes. A concentrated solution of dihydrazine succinate in an aqueous methanol mixture (50/50 vol) was added to the microemulsion and allowed to react for 3 hours to crosslink the aminooxy moiety using aldehyde functional groups. The resulting nano article was purified by precipitation and resuspended in an aqueous solution in the presence of excess dihydrazine succinate. The reaction of dihydrazine succinate (with the remaining aldehyde on the nanoarticle) was allowed to proceed for 12 hours.

Example 16: Doxorubicin (Dox) Binding via Amide Linkage
Doxorubicin (120 mg) was completely dissolved in 15.0 mL water and 15.0 mL HEPES buffer (pH 7.5 (0.20 M)) was added. To a solution of the nanoarticle of Example 8 (1.0 g) dissolved in 10.0 mL HEPES buffer (pH 7.5 (0.20 M)), 10.0 mL of water was added. To this solution was added 160 mg N-hydroxysuccinimide (NHS) and 50 mg 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) successively. Twenty minutes after EDC addition, 7.5 mL of doxorubicin solution was added. A second 50 mg aliquot of EDC was added followed by a second amount of doxorubicin, delayed by 20 minutes. After an additional 0.5 hour reaction interval, the addition / delay cycle was repeated two more times so that the entire doxorubicin solution was added. The reaction mixture was then stirred for 24 hours. Unreacted starting materials and by-products were separated from the Dox-nanoarticle product by passing through a HiPrep 26/10 desalting column and the nanoarticle was isolated as a dry solid by lyophilization. The Dox loading of the resulting article was about 6-8% by weight. The doxorubicin content of the nanoarticle can be altered by using a small amount of this material during the addition or by reducing the total number of reaction cycles.

(Example 17: doxorubicin binding by hydrazone ligation (high filling))
1.0 g of material from Example 9 is dissolved in 40 mL of 0.1 M ammonium formate (pH 8.5) and excess carbohydrazide (100 equivalents of carbohydrazide for each diacetone acrylamide in the starting material). To the concentrated solution. The reaction was allowed to proceed for 12 hours at 4 ° C. In this way, a hydrazone bond was formed between the carbohydrazide and the ketone portion of the nanoarticle. The reaction mixture was purified from excess carbohydrazide using dialysis or size exclusion chromatography.

  200 mg of doxorubicin (diluted in 100 mL water) was added to the carbohydrazide functionalized scaffold in water buffered with ammonium formate. The reaction was allowed to proceed for 18-24 hours at 4 ° C. In this way, a hydrazone bond was formed between the ketone moiety of doxorubicin and the carbohydrazide linked to the nanoarticle. The resulting nanoarticle with covalently bound doxorubicin was then purified from unreacted doxorubicin and by-products using dialysis or size exclusion chromatography.

(Example 18: Doxorubicin binding by hydrazone ligation (low filling))
1.0 g of material from Example 9 is dissolved in 40 mL of 0.1 M potassium borate (pH 8) and excess carbohydrazide (10 equivalents of carbohydrazide for each diacetone acrylamide in the starting material). To the concentrated solution. The reaction was allowed to proceed for 1 hour. The reaction mixture was purified from excess carbohydrazide using dialysis or size exclusion chromatography. In this way, a hydrazone bond was formed between the carbohydrazide and the ketone portion of the nanoarticle.

  50 mg of doxorubicin (diluted in 20 mL water) was added to the carbohydrazide functionalized nanoarticle in water buffered with ammonium formate. The reaction was allowed to proceed for 1-6 hours. In this way, a hydrazone bond was formed between the ketone moiety of doxorubicin and the carbohydrazide linked to the nanoarticle. The resulting nanoarticle with covalently bound doxorubicin was then purified from unreacted doxorubicin and by-products using dialysis or size exclusion chromatography.

(Example 19: Cyclosporine bond by amide bond)
Cyclosporine was mixed with 4-benzoylbenzoic acid (BBa) in benzene at a molar ratio of 1-2. This solution was purged with nitrogen gas and photolyzed at room temperature at a wavelength of 320 nm (US Pat. No. 5,405,785). After photolysis, benzene was evaporated on a rotary evaporator under reduced pressure and the dried product was dissolved in methanol. The product was isolated by preparative HPLC. Cyclosporine-BBa was then added to a methanol solution of N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (approximately 1 to 1.2 to 1 molar ratio). The reaction was allowed to proceed overnight at room temperature. The formation of activated esters can be detected using a neutral Fe-hydroxamate test. Cyclosporine-BBa-NHS can be attached to a nanoarticle scaffold composed of an amine moiety (the preparation of which is described in Example 13) using EDC coupling.

(Example 20: Dexamethasone binding)
Caliciamycin binding via hydrazone binding)
The synthesis of the calithiamycin derivative (N-acetyl-γ carotimycin dimethyl hydrazide) has been reported previously (Upeslacis J. et al., Cancer Res., 1993, 53, 3336-3342). 1.0 g of IMMA-CiBA-DAA scaffold nanoarticle is dissolved in 40 mL of 0.1 M ammonium formate (pH 8.5) and excess N-acetyl-γ calicheamicin dimethyl hydrazide (each diester in the starting material). To a concentrated solution of 100 equivalents of N-acetyl-γ-calyciamycin dimethyl hydrazide to acetone acrylamide). The reaction was allowed to proceed for 12 hours at 4 ° C. In this way, a hydrazone bond was formed between N-acetyl-γ carotimycin dimethyl hydrazide and the ketone portion of the nanoarticle. The reaction mixture was purified from excess N-acetyl-gamma-calyciamycin dimethyl hydrazide using a 26/10 desalting column.

(Example 21: Dexamethasone binding)
Dexamethasone attachment to the nanoarticle is possible through a glycine functionalized linker. The linker compound is prepared by using glycine functionalized 2-CI Trityl resin (2002/2003 NovaBiochem Catalog, page 2.16-2.17, CalBiochem-NovaBiochem Corp., San Diego, USA). The acid portion of the glycine linker is reacted with the 21 carbon hydroxy group of dexamethasone using carbodiimide. Excess dexamethasone is used to react with all acid groups of the glycine linker. The resulting compound is cleaved at the amine site using 1% trifluoroacetic acid (TFA). This produces a free amine that can be conjugated with an acid (NaA) functionalized nanoarticle. The product is purified by use of HiPrep 26/10 desalting column / size exclusion chromatography. The ester bond is then cleaved in vivo to release dexamethasone.

(Example 22: Incorporation of gemcitabine)
The nanoarticle of Example 9 (1.0 g) (preferably containing DAA or oxidized sugar) is dissolved in 10.0 mL of PBS buffer (pH 7.2, 0.2 M). 15.5 mg of gemcitabine is added to the nanoarticle solution (35 ° C.). The reaction is allowed to proceed for 1 hour, after which the nano article is isolated by size exclusion chromatography. The aqueous nanoarticle can be lyophilized at this point.

Example 23: Binding of methotrexate
Methotrexate (100 mg, 0.22 mmol) was added to boc protected ethanolamine (100 mg, 0.44 mmol), dicyclohexylcarbodiimide (DCC) (91 mg, 0.44 mmol) and 4-dimethylaminopyridine (DMAP) (36 mg, 0.293 mmol). Dissolve in dimethyl sulfoxide (DMSO) (10 mL) at 0 ° C. The reaction mixture is left overnight at room temperature. The N-protected aminoester methotrexate is washed with 0.1N HCl, dried and evaporated under reduced pressure to give a solid product. The boc protecting group is removed by dissolving the product in a 50:50 mixture of methylene chloride and TFA and stirring at room temperature for 3 hours. Evaporating the solvent gives the deprotected amino ester derivative (Minko T. et al., Cancer Chemother. Pharmacol., 2002, 50, 143-150).

  The aminoester derivative dry powder (100 mg) is completely dissolved in 2 mL DMSO. A solution (0.20 M) of the nanoarticle of Example 8 (1.0 g) dissolved in 10.0 mL HEPES buffer (pH 7.5) is added to 10.0 mL water. To this solution is added methotrexate solution and then 160 mg N-hydroxysuccinimide (NHS) and 50 mg 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) are added sequentially. After 30 minutes, a second 50 mg aliquot of EDC is added. After an additional 0.5 hour reaction interval, the addition / delay cycle is repeated two more times. The reaction mixture is then stirred for 24 hours. Unreacted starting materials and by-products are isolated from the methotrexate-nano article product by passing through a HiPrep 26/10 desalting column, and the nano article is isolated as a dry solid by lyophilization.

(Example 24: Binding of salicylic acid)
400 mg of succinic anhydride is reacted with 2.0 g of IMMA (prepared according to Example 1) in pyridine to a level of about 1 carboxylic acid moiety per 3 sugar repeats of insulin and Isolate according to the method described in 1. Then 1.0 g of this acid modified IMMA (AM-IMMA) is coupled with salicylic acid (300 mg) in DMF using 1,3-dicyclohexylcarbodiimide (DCC, 500 mg). In this procedure, the AM-IMMA is dissolved in anhydrous DMF (50 mL) and DCC is added. After 4 hours, salicylic acid is added and the reaction is stirred overnight. The next day, the reaction is filtered and the DMF is removed under reduced pressure. The crude material is dissolved in water (50 mL), filtered again, and the product is isolated after lyophilization. This salicylic acid modified SAM-IMMA is then used in place of the unmodified IMMA in the formulation procedure described in Example 8 (synthetic and isolation procedures are not changed) to give SAM-IMMA / CiBA / acrylic acid. Produce a sodium nanoarticle framework.

(Example 25: Binding of camptothecin)
20-O-peptidyl-camptothecin (120 mg) (prepared according to the procedure of Farao M. et al. (Molecular Cancer Therapeutics, 2003, 2, 29-40)) was dissolved in 15 mL DMSO and 15.0 mL HEPES buffer Solution (pH 7.5, 0.20 M) was added. To a solution (0.20M) of nanoarticle (1.0 g) (acid-containing nanoarticle) made according to Example 8 dissolved in 10.0 mL HEPES buffer (pH 7.5), 10.0 mL of water was added. Added. To this solution, 160 mg of N-hydroxysuccinimide (NHS) and 50 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were added sequentially. Twenty minutes after the addition of EDC, 7.5 mL of 20-O-peptidyl-camptothecin solution was added. A second 50 mg aliquot of EDC was added and then after a 20 minute delay, a second amount of 20-O-peptidyl-camptothecin was added. After an additional 0.5 hour reaction interval, the addition / delay cycle was repeated two more times, ensuring the addition of all 20-O-peptidyl-camptothecin solution. The reaction mixture was then stirred for 24 hours. Unreacted starting materials and by-products are separated from the 20-O-peptidyl-camptothecin nanoarticle product by passing through a HiPrep 26/10 desalting column and the nanoarticle is dried solid by lyophilization. As isolated.

(Example 26: Malonate-containing nano article and N, O complexed cis-diaminoplatinum)
In a solution of the nano article (1.0 g) described in Example 11 (composed of 80 wt% buffer, 14 wt% IMMA, 6 wt% MalAc and eosin Y) in 0.1 M sodium nitrate (pH 7.4). A 50 mM solution of cis-diamine diaquaplatinum dinitrate in sodium nitrate (26 mL) was added. The reaction is allowed to proceed for 12 hours at room temperature. The use of nitrate is advantageous for ligand exchange. The initially formed O, O-platinum-malonate complex can rearrange itself to a more stable O, N-malonate complex (this transformation can be followed by Pt NMR). The unreacted platinum derivative is then separated from the platinum complexed nanoarticle product by passing through a HiPrep 26/10 desalting column. The nanoarticle is isolated as a dry solid by lyophilization.

Example 27: cis-diamino-platinum complexed with amide and acid moieties of CIBA and N, O
To a solution of 1.0 g nanoarticle (composed of 80 wt% buffer, 14 wt% IMMA, 6 wt% CiBA and eosin Y) in 0.1 M sodium nitrate (pH 7.4), cis- A 100 mM solution (18 mL) of diaminodiaquaplatinum dinitrate was added. The reaction was allowed to proceed overnight at room temperature. Unreacted platinum derivatives and too complex platinum derivatives were separated from the platinum complexed nanoarticle product by passing through a HiPrep 26/10 desalting column. The nanoarticle was isolated as a dry solid by lyophilization.

Example 28: Targeted cis-platin nanoarticle (post-targeting load)
Nanoarticles made according to Example 12 (82 wt% buffer, 14 wt% IMMA, 2 wt% CIBA and 2 wt% N-methacryloylated Gly-Gly peptide (made according to US Pat. No. 5,037,883), and eosin Y Is prepared according to the procedure described in Example 30 (DTT reduction), functionalized with PEG dBA (Example 30), and ErbB-2 ligand (bromo) as described in Example 37. Targeted with acetamide functionalized cyclic F [CDGFYAC] YMDV).

  To a solution of these nanoarticles (1.0 g) in 0.1 M sodium nitrate (pH 7.4) was added a 100 mM solution of cis-diamine diaquaplatinum dinitrate (18 mL) in sodium nitrate. The reaction was allowed to proceed overnight at room temperature. Unreacted platinum derivatives and too complex platinum derivatives were separated from the platinum complexed nanoarticle product by passing through a HiPrep 26/10 desalting column. The nanoarticle was isolated by lyophilization.

Example 29: Binding of 5-fluorouracil (5FU)
The chloromethyl ester of Boc-protected glycine is coupled to 5FU according to the method of Taylor et al. (Taylor HE, Sloan KB Journal of Pharmaceutical Sciences, 1998, 87, 15). After deprotection with trifluoroacetic acid (ATF) and removal of excess TFA under reduced pressure, amine functionalized 5FU (made from 198 mg of Boc-protected starting material) was immediately added for attachment to carboxylic acid-containing nanoarticles. use. This material is completely dissolved in 15.0 mL water and 15.0 mL HEPES buffer (pH 7.5, 0.20 M, 15.0 mL) is added. To a solution of the nanoarticle of Example 8 (0.20 M) dissolved in 10.0 mL HEPES buffer (pH 7.5), 10.0 mL of water is added. To this solution is added successively 160 mg N-hydroxysuccinimide (NHS) and 50 mg 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC). Twenty minutes after the addition of EDC, 7.5 mL of amine functionalized 5FU solution was added. The resulting solution is gently stirred for 0.5 hours. A second 50 mg aliquot of EDC is added followed by a second amount of derivatized 5FU after a 20 minute delay. After an additional 0.5 hour reaction interval, this addition / delay cycle is repeated two more times, thus ensuring the addition of all derivatized 5FU solution. The reaction mixture is then stirred for 24 hours. Unreacted starting material and by-products are separated from the nanoarticle product by passing through a HiPrep 26/10 desalting column and the nanoarticle is isolated as a dry solid by lyophilization.

(Example 30: Linker binding)
A doxorubicin-loaded article (400 mg) as described in Example 16 is added to 0.5 mL of phosphate buffer (pH = 7.2, 0.10 M, 0.15 M NaCl, this buffer is often referred to as PBS. Dissolved). To this solution was added dithiothreitol (DTT, 198 mg) and the reduction reaction was gently stirred for 2.0 hours. The resulting article was analyzed by using a high performance liquid chromatography (FPLC) system fitted with a HiPrep 26/10 desalting column using 100 mM phosphate buffer (pH = 8.0) as the eluting solvent. Separated from material. PEG-1500 dBA (2.59 g) was weighed into a 50 mL vial. The freshly purified nanoarticle solution was added to the PEG-1500 dBA and the PEG coupling reaction was gently stirred under aluminum foil for 2 hours at room temperature. The nanoarticle was then separated from the other components of the reaction mixture using the same FPLC configuration described above. The pooled fractions containing the nanoarticles were used for the next procedure described here in which the specific targeting element binds.

(Example 31: Binding of BMPEO ₄ )
The doxorubicin-loaded article (400 mg) described in Example 16 was dissolved in PBS. To this solution was added dithiothreitol (DTT, 198 mg) and the reduction reaction was gently stirred for 2.0 hours. This article was separated from other materials by using FPLC fitted with a HiPrep 26/10 desalting column using PBS as the eluting solvent. Homobifunctional crosslinker 1,11-bis-maleimidotetraethylene glycol (BMPEO ₄ , 379 mg) was added to the pooled FPLC fraction containing the nanoarticles. The reaction was allowed to proceed for 2 hours at room temperature with stirring. Unreacted linker was then removed from the nanoarticle using the same FPLC described above. These materials are used in a manner similar to PEG-dBA functionalized articles.

Example 32: Binding of erbB1 ligand to Dox loaded article
Disulfide bridged cyclic peptides:

（１７４ｍｇ）を、実施例１６に記載されるナノ物品を含むＦＰＬＣから溶離されたプールされた画分に添加した。この反応物を、アルミニウム箔下で、２時間、穏やかに攪拌した。この反応混合物中の他の成分からのナノ物品の分離を、溶媒として脱イオン水を使用するＨｉＰｒｅｐ２６／１０脱塩カラムを取り付けたＦＰＬＣを使用することによって達成した。凍結乾燥によって、ナノ物品生成物を単離し得る。

(174 mg) was added to the pooled fraction eluted from the FPLC containing the nanoarticle described in Example 16. The reaction was gently stirred for 2 hours under aluminum foil. Separation of the nanoarticles from other components in the reaction mixture was achieved by using FPLC fitted with a HiPrep 26/10 desalting column using deionized water as solvent. The nanoarticle product can be isolated by lyophilization.

Example 33: Binding of erbB2 ligand to Dox-loaded article
Disulfide bridged cyclic peptides:

（記号ｄＦは、フェニルアラニンのＤ異性体を表わし、そしてｄＹはチロシンのＤ異性体を表わす、１６２ｍｇ）を、実施例１６に記載されるナノ物品を含むＦＰＬＣから溶離されたプールされた画分に添加した。この反応物を、アルミニウム箔下で、２時間穏やかに攪拌した。この反応混合物中の他の成分からのナノ物品の分離を、溶媒として脱イオン水を使用するＨｉＰｒｅｐ２６／１０脱塩カラムと取り付けたＦＰＬＣを使用することによって達成した。凍結乾燥によって、このナノ物品生成物を単離し得る。

(The symbol dF represents the D isomer of phenylalanine and dY represents the D isomer of tyrosine, 162 mg) in the pooled fraction eluted from the FPLC containing the nanoarticle described in Example 16. Added. The reaction was gently stirred for 2 hours under aluminum foil. Separation of the nanoarticles from other components in the reaction mixture was accomplished by using an FPLC attached with a HiPrep 26/10 desalting column using deionized water as the solvent. The nanoarticle product can be isolated by lyophilization.

Example 34: Binding of VEGFR2 ligand to Cox loaded article
The peptide ATWLPPRC (120 mg) was added to the pooled fraction eluted from the FPLC containing the nanoarticle described in Example 16. The reaction was gently stirred for 2 hours under aluminum foil. Separation of the nanoarticles from other components in the reaction mixture was accomplished by using an FPLC attached with a HiPrep 26/10 desalting column using deionized water as the solvent. The nanoarticle product can be isolated by lyophilization.

Example 35: RGD attachment to PEG dBA functionalized articles
Cyclic RGD peptide (HAP3C, RGDdFC, or FW 576) (62 mg) was added to doxorubicin-containing nanoarticle (355 mg) with conjugated PEG ₄₀₀ dBA linker tether (Example ₄₀₀ using PEG ₄₀₀ dBA according to the procedure of Example 30). To the solution containing (prepared by functionalizing the Dox article) at 10 mg / mL in PBS (pH 8). The reaction was allowed to proceed for 2 hours, after which 13 mg cysteine was added. The reaction with cysteine is allowed to proceed for 1 hour, after which the nanoarticle is purified by FPLC using volatile buffer (pH 7-9) as eluent to have doxorubicin with PGD attached via PEG ₄₀₀ dBA chain A containing nano article was obtained. The nanoarticle was lyophilized.

Example 36: Binding of erbB1 ligand to PEG dBA functionalized material
100 mg of cyclic ErB1 peptide with cysteine at the N-terminus (CYCPIWKFPDECYY) was added to 300 mg of doxorubicin-containing nanoarticle conjugated with PEG ₄₀₀ dBA linker tether (Dox- of Example 17 according to the procedure of Example 30) The material was prepared by functionalizing with PEG ₄₀₀ dBA) at 5 mg / mL to a solution containing in PBS (pH 8). The reaction was carried out for 4 hours, after which 11 mg cysteine was added. Reaction with cysteine was performed for 1 hour, and then the nano article was purified by FPLC to obtain a doxorubicin-containing nano article having Erb1 bound thereto via a PEG ₄₀₀ dBA chain.

Example 37: Conjugation of erbB2 ligand to PEG dBA functionalized material
50 mg of cyclic ErB1 peptide (CdFCDGFdYACYMDV) with cysteine at the N-terminus was pre-dissolved in water and 300 mg of doxorubicin-containing nanoarticle conjugated with PEG ₄₀₀ dBA linker tether (according to the procedure of Example 30). Dox-material was prepared by functionalizing with PEG ₄₀₀ dBA) at a final concentration of 2.5 mg / mL to a solution containing in PBS (pH 8). The reaction was carried out for 4 hours, after which 11 mg cysteine was added. The reaction was performed for 2 hours, and then the nano article was purified by FPLC to obtain a doxorubicin-containing nano article having Erb2 bound thereto via a PEG ₄₀₀ dBA chain.

Example 38: Binding of VEGFR-2 ligand to PEG dBA functionalized material
Cysteine-terminated VEGFR-2 peptide (ATWLPPRC) (100 mg) was added to 355 mg of doxorubicin-containing nanoarticle conjugated with PEG ₄₀₀ dBA linker tether (Example 17 Dox-substance was converted to PEG ₄₀₀ according to the procedure of Example 30). (prepared by functionalizing with dBA) was added at 10 mg / mL to a solution containing in PBS (pH 8). The reaction was run for 2 hours, after which 13 mg cysteine was added. The reaction was carried out for 1 hour, and then the nano article was purified by FPLC to obtain a doxorubicin-containing nano article having VEGFR-2 bound thereto via a PEG ₄₀₀ dBA chain.

Example 39 Tolerability of Hydrogel Skeleton in Mice
A framework nanoarticle of 14% IMMA and 1 wt% DAA was prepared according to the procedure of Example 9 omitting the addition of CiBA.

Before the start of the experiment, 9 female SCID / Rag 2M mice (9 weeks old, body weight 18-22 g) were randomly divided into 3 groups of 3 animals. The substance was solubilized in saline and injected intravenously into the tail vein of mice. Intravenous administration of nanoarticles at 100 mg / kg, 200 mg / kg and 400 mg / kg was well tolerated with no acute or delayed signs of toxicity. Body weight was monitored over 16 days. There was no weight loss during this period. The results are shown in Table 1:
(Table 1: Average body weight change (%) in mice (n = 3) after a single intravenous injection of nanoarticles)

（実施例４０：ＦＩＴＣ標識化ＮＰマトリックスのＰＫ）
ＦＩＴＣ標識化ナノ粒子（ＮＰ）またはＰＥＧ−４００と結合したＦＩＴＣ標識化ナノ粒子（ＮＰ−ＰＥＧ）を、実施例８に記載の逆ミクロエマルジョンポリマー化法を用いて作製し、ここで、水相は、８３重量％の水、１４重量％ ＩＭＭＡ、１重量％ ＡＰＭＡ、および１重量％ ＮＯＢＡから構成されていた。フルオレセインイソチオシアネート（ＦＩＴＣ）を、フルオレセインのイソチオシアネートとナノ粒子上のアミン含有部分のアミノプロピルメタクリレート（ＡＰＭＡ）との間の結合を介して結合した。

(Example 40: PK of FITC-labeled NP matrix)
FITC-labeled nanoparticles (NP) or FITC-labeled nanoparticles conjugated with PEG-400 (NP-PEG) were made using the reverse microemulsion polymerization method described in Example 8, where the aqueous phase Consisted of 83 wt% water, 14 wt% IMMA, 1 wt% APMA, and 1 wt% NOBA. Fluorescein isothiocyanate (FITC) was coupled through a bond between fluorescein isothiocyanate and the amine-containing moiety of aminopropyl methacrylate (APMA) on the nanoparticles.

  Female Balb / c mice were injected intravenously with 50 mg / kg FITC labeled nanoparticles (NP) or FITC labeled nanoparticles conjugated with PEG-400. Treatment groups were evaluated after 15 minutes, 2 hours, 6 hours, and 24 hours. Blood and tissue samples were collected at the time points described above. Tissue samples were quickly frozen before further analysis. Plasma was prepared from blood samples through centrifugation at 1500 g for 10 minutes. Plasma and tissues were assayed for the presence of NP or NP-PEG through a fluorescence assay using a microplate fluorescence reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Plasma samples were diluted 10-fold with PBS. The final assay volume was 100 psi per well of a black bottom 96 well plate. Tissues were extracted using a tissue homogenizer in 2 ml of PBS containing 0.05% Triton X-100. The tissue sample was then diluted 5-fold in PBS to a capacity of 100 psi in a black bottom 96 well plate.

  Plasma removal of FITC-NP in μg of particles resulted in a long half-life (6.6 hours) in blood for both bare (unfunctionalized) and PEG-400 conjugated nanoparticles. Indicated. Biodistribution was analyzed by measuring fluorescence in the liver, spleen, kidney, heart and lung. Low accumulation in the liver and spleen was detected for both types of nanoparticles from tissue extracts taken at 4 time points over 24 hours. Nanoparticles were found in the lung, heart and kidney.

  (Table 2: Clearance of FITC-labeled NP from whole blood compartment during 24 hours. Total amount of injected nanoparticles per mouse was 1000 μg (normalized to 20 g body weight). The value is μg / tissue.)

（実施例４１：ＲＧＤ−Ｄｏｘ−ＮＰのＣＡＭ結果）
７日齢のニワトリ胚の絨毛尿膜（ＣＡＭ）を、マウス乳癌ＣＩ−６６を各１０^５細胞と共に、２４時間インキュベートし、その後、５μｇのドキソルビシン、５μｇのナノ粒子中のＤｏｘ等価物（１４／１／１ ＩＭＭＡ／ＮＯＢＡ／ＮａＡ、ＰＥＧ_３４００）、５μｇの環状ＲＧＤで官能化したナノ粒子中のＤｏｘ等価物（１４／１／１ ＩＭＭＡ／ＮＯＢＡ／ＮａＡ、ＰＥＧ_３４００−ｃＲＧＤ）、またはＰＢＳコントロールのいずれかを含むディスク（ｄｉｓｋ）と共にインキュベートした。ナノ粒子に結合したリンカーを、実施例３１で記載のように作製した。

(Example 41: CAM result of RGD-Dox-NP)
Seven-day-old chick embryo chorioallantoic membrane (CAM) was incubated with 10 ⁵ cells each of mouse breast cancer CI-66 for 24 hours, after which the Dox equivalent in 5 μg doxorubicin, 5 μg nanoparticles (14 / 1/1 IMMA / NOBA / NaA, PEG ₃₄₀₀ ) Dox equivalent in nanoparticles functionalized with 5 μg cyclic RGD (14/1/1 IMMA / NOBA / NaA, PEG ₃₄₀₀ -cRGD), or PBS control Incubated with discs containing either. Linkers attached to the nanoparticles were made as described in Example 31.

  Three days later, the CAM was excised and photographed for analysis. Doxorubicin alone was most effective in reducing the number of blood vessels. However, the application was also the most toxic, so 6 out of 10 CAMs in this group were necrotic and 3 out of 10 embryos in the group died during the treatment. Dox-NP worked well in changing blood vessel distribution around the disc. Dox-NP-cRGD was more effective at reducing the number of blood vessels around the disc compared to Dox-NP.

Example 42: In vivo toxicity / efficacy data for RGD-Dox NP and Dox NP
The potency and toxicity of nanoarticle-bound doxorubicin (Dox-NP) was compared to doxorubicin (Dox) and saline control. Composition 14/1/1 IMMA / CiBA / NaA Dox-NP, 5.6% Dox was made according to the following procedure in Example 16.

CL66 breast cancer cells are grown to 80-90% confluence in enriched medium, trypsinized with 1 × trypsin for 2 minutes at 37 ° C., rinsed with Hanks balanced salt solution (no calcium and magnesium), It was then resuspended at 1 × 10 ⁶ cells / ml and 0.1 ml was injected into the fat pad of the female Balb / mouse breast.

  Lyophilized Dox-NP was reconstituted at the appropriate concentration in sterile PBS and stored at 2-8 ° C. for at least 1 hour or overnight. Excipient Dox was dissolved at the appropriate concentration in PBS. 200 μL was injected intravenously via the tail vein of mice 13 days, 15 days and 18 days after tumor induction. Dox was injected at 225 μg / animal while Dox-NP was injected at nanoparticle levels sufficient for Dox delivery at 250 μg / animal, 420 μg / animal and 560 μg / animal.

  Tumor volume was measured superficially in two dimensions. Weight loss (or gain) and survival were recorded. A significant decrease in tumor size was observed in the 250 μg / animal group. Nanoparticles containing higher doses (eg, Dox 225 μg / animal dose) of Dox were highly toxic, but mice injected with Dox had slightly longer survival times. The lowest dose of Dox-NP had therapeutic activity and slowed the tumor growth rate, thus making the tumor smaller. At death, tumors were smaller in the 250 μg / animal Dox-NP group than in the Dox group, and their body weight was lower than in the Dox group. However, the smaller the tumor, the smaller the survival difference (after tumor injection) between 250 μg / animal Dox-NP mice and control mice. This suggests that Dox-NP is active and is counteracted by continuous tumor growth in the absence of further treatment.

Example 43: In vivo toxicity / efficacy data for RGD-Dox NP and Dox NP
The efficacy and toxicity of nanoarticle-bound doxorubicin (Dox-NP and Dox-NP-cRGD) was studied. Composition 14/1/1 IMMA / CiBA / NaA Dox-NP, 5.6 wt% Dox, PEG ₄₀₀ (with and without cRGD) was made according to the procedure of Example 16. PEG ₄₀₀ dBA was coupled according to the procedure of Example 30. Tumor-bearing mice were inoculated, treated and observed as described in Example 42 (above). Intravenous injections were given three times a week in the first week with 1200 μg Dox equivalents (Dox-NP and Dox-NP-cRGD). Only one mouse in the Dox-NP group died and all mice in the Dox-NP-cRGD group survived. Average tumor volume increased more than 30 times in the saline control group. In contrast, the Dox-NP group increased 12-fold and the Dox-NP-cRGD group increased 6-fold. Large tumors in the Dox-NP-cRGD group showed significant necrosis.

  (Table 3: Change in tumor size (n = 5) expressed as mean x-fold increase in volume after treatment (days after treatment (tiw)))

（ＮＡ＝適用不能）
（実施例４４：Ｄｏｘ−アミド結合ナノ粒子のインビトロ細胞毒性）
ドキソルビシンは、共有結合（細胞内環境において非常にゆっくり分解されるかまたはそれより迅速に分解される）からなる結合を介してナノ物品骨格に結合し得る。例えば、アミド結合はよりゆっくりと分解されるが、他の結合（例えば、ヒドラゾン結合からなる結合）は、加水分解を介してより迅速に分解される。アミド結合を介してナノ粒子に結合するＤｏｘの毒性を、インビトロ毒性アッセイにおいて、Ｃ−３２細胞（ヒト黒色腫）に対して試験した。組成物１４／２／１ＩＭＭＡ／ＣｉＢＡ／ＮａＡ のＤｏｘ−ナノ−物質を、実施例８に記載の手順に従って作製した。

(NA = not applicable)
Example 44: In vitro cytotoxicity of Dox-amide bonded nanoparticles
Doxorubicin can be bound to the nanoarticle backbone via a bond consisting of a covalent bond (degraded very slowly or more rapidly in the intracellular environment). For example, amide bonds are degraded more slowly, while other bonds (eg, bonds consisting of hydrazone bonds) are more rapidly degraded via hydrolysis. The toxicity of Dox binding to nanoparticles via an amide bond was tested against C-32 cells (human melanoma) in an in vitro toxicity assay. A Dox-nano-material of composition 14/2/1 IMMA / CiBA / NaA was made according to the procedure described in Example 8.

In a 96-well tissue culture microtiter plate, 10,000 C-32 cells per well in culture medium were allowed to bind for 2 hours and then incubated with varying concentrations of Dox-amide nano-material and free Dox. . Cells were incubated for 48 hours in a humid chamber at 37 ° C., 5% CO ₂ . Cell proliferation was assayed using a colorimetric MTT kit (CT01, Chemicon, Int.). 50% growth inhibition was determined compared to 0.8 μg / mL free Dox versus 0.3 μg / mL of the Dox equivalent.

Example 45: In vitro toxicity of Dox-hydrazone binding nanoparticles
The toxicity of Dox binding to nanoarticles via hydrazone binding was tested in an in vitro toxicity assay against C-32 cells (human melanoma). Composition 14/2/1 IMMA / CiBA / DAA Dox-nano-material was made according to the procedure described in Example 9.

In a 96-well tissue culture microtiter plate, 10,000 C-32 cells per well in culture medium were allowed to bind for 2 hours and then incubated with varying concentrations of Dox-hydrazone nano-material and free Dox. did. Cells were incubated for 48 hours in a humid chamber at 37 ° C., 5% CO ₂ . Cell proliferation was assayed using a colorimetric MTT kit (CT01, Chemicon, Int.). 50% growth inhibition was determined relative to 0.07 μg / mL of the Dox equivalent compared to 0.8 μg / mL free Dox.

Claims (32)

A polyhydrogel nano article comprising:
i) a polymer scaffold comprising a crosslinked hydrophilic building block;
ii) a therapeutic agent covalently attached to the polymer scaffold; and iii) two or more recognition elements covalently attached to the polymer scaffold;
A hydrogel nano article comprising:   The polyhydrogel nanoarticle of claim 1, wherein the recognition element is directly covalently bound to a polymer molecule of the scaffold.   The polyhydrogel nanoarticle of claim 1, wherein the recognition element is covalently attached to a polymer molecule of the scaffold by a linker molecule.   The hydrogel nano article according to claim 3, wherein the linker molecule is a polyethylene glycol chain.   The hydrogel nano article according to any one of claims 1 to 4, wherein at least one of the recognition elements has an amino acid sequence RGD.   The hydrogel nanoarticle according to any one of claims 1 to 4, wherein at least one of the recognition elements is a growth factor EGF.   The hydrogel nanoarticle of any one of claims 1-6, wherein the scaffold comprises at least some degradable covalent bonds.   The hydrogel nano article according to any one of claims 1 to 7, wherein the hydrophilic building block further comprises a low-molecular crosslinking agent.   9. A hydrogel nano article according to any one of the preceding claims, wherein at least some of the hydrophilic building blocks are carbohydrate based monomers.   10. The hydrogel nanoarticle of claim 9, wherein at least some of the carbohydrate based monomers are functionalized with an acrylate, moiety, methacrylate moiety, acrylamide moiety, or methacrylamide moiety.   11. A hydrogel nano article according to claim 9 or 10, wherein at least some of the hydrophilic building blocks comprise inulin or dextran.   12. The hydrogel nanoarticle of claim 11, wherein at least some of the hydrophilic building blocks are inulin multimethacrylate.   13. The hydrogel nanoarticle according to any one of claims 1 to 12, wherein at least some of the hydrophilic building blocks are N, N'-cystine bisacrylamide.   13. A hydrogel nano article according to any one of claims 1 to 12, wherein at least some of the hydrophilic building blocks are diacetone acrylamide.   13. A hydrogel nano article according to any one of the preceding claims, wherein at least some of the hydrophilic building blocks are aminopropyl methacrylamide.   16. A hydrogel nanoarticle according to any one of claims 1 to 15, wherein the building block comprises inulin multimethacrylate, N, N'-cystine bisacrylamide, and sodium acrylate.   16. The hydrogel nano article of any one of claims 1-15, wherein the building block comprises inulin multimethacrylate, N, N'-cystine bisacrylamide, and diacetone acrylamide.   18. A hydrogel nano article according to any one of the preceding claims, further comprising at least one polyethylene glycol molecule covalently bonded to the polymer matrix.   The hydrogel nano article according to any one of claims 1 to 19, wherein the therapeutic agent is a chemotherapeutic agent.   20. The hydrogel nano article of claim 19, wherein the therapeutic agent is doxorubicin or a doxorubicin analog.   21. The hydrogel nano article of any one of claims 1-20, wherein the therapeutic agent is bound to the scaffold via a hydrolyzable bond. The following formula I:

Of the compound N, N′-cystine bisacrylamide. Formula IV below:

The compound inulin multimethacrylate, wherein n is from about 5 to about 50. A method for controllably releasing a therapeutic agent into an environment within a mammalian body, the method comprising administering a hydrogel nanoarticle to the environment, the hydrogel nanoarticle comprising:
i) a polymer scaffold comprising a crosslinked hydrophilic building block; and ii) a therapeutic agent covalently attached to the scaffold;
Including a method. A method for controlled delivery of a therapeutic agent to the vicinity of a targeted cell or tissue type, the method comprising placing a hydrogel nanoarticle into an environment comprising the targeted cell or tissue type. Comprising the step of administering, wherein the hydrogel nano article comprises:
i) a polymer scaffold comprising a crosslinked hydrophilic building block;
ii) a therapeutic agent covalently bound to the scaffold; and iii) two or more recognition elements covalently bound to the scaffold, wherein the recognition element is expressed in the targeted cell or tissue type A recognition element having binding affinity for the molecule,
Including a method.   26. Use of the method according to claim 24 or 25 for the treatment of cancer. A method for synthesizing a hydrogel recognition element functionalized polymer nanoarticle comprising the following:
Forming a polymer scaffold of the nanoarticles via crosslinking of hydrophilic building blocks in a discontinuous aqueous phase of the inverse microemulsion, wherein at least some of the building blocks are N, N′-cystine bis A process which is acrylamide;
Reducing the polymer scaffold to generate free thiols from disulfide bonds of the N, N′-cystine bisacrylamide;
Adding a linker molecule to attach the linker to the polymer scaffold, the linker molecule containing a group reactive with a thiol; and adding a recognition element comprising the steps of: A recognition element comprising a group reactive with the free end of the linker molecule,
Which results in a recognition element functionalized nanoarticle. A method for synthesizing a hydrogel recognition element functionalized polymer nanoarticle comprising the following:
Forming a polymer scaffold of the nanoarticles via crosslinking of hydrophilic building blocks in a discontinuous aqueous phase of the inverse microemulsion, wherein at least some of the building blocks are N, N′-cystine bis A process which is acrylamide;
Reducing the polymer scaffold to generate a free thiol from the disulfide bond of the N, N′-cystine bisacrylamide; and adding a linker molecule to a recognition element functionalized linker attached to one end of the linker molecule Attaching a molecule to the polymer scaffold, the linker molecule comprising the recognition element, the linker molecule comprising a group reactive with a thiol,
Which results in a recognition element functionalized nanoarticle.   29. A method according to claim 27 or 28, wherein at least some of the hydrophilic building blocks are carbohydrate based monomers.   Prior to reducing the polymer scaffold, the method further comprises adding a therapeutic agent to the nanoarticle, wherein the therapeutic agent is reactive with the polymer scaffold and a group that covalently attaches the therapeutic agent to the scaffold. 30. A method according to claim 27, 28 or 29. A hydrogel nano article comprising:
i) a polymer scaffold comprising a crosslinked hydrophilic building block; and ii) a therapeutic agent covalently bonded to the polymer scaffold;
A hydrogel nano article comprising: In addition:
iii) two or more recognition elements covalently bound to the polymer scaffold, the recognition elements having binding affinity for biomolecular structures expressed in specific cells or specific tissues element,
32. The hydrogel nanoarticle of claim 31, comprising: